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Lignocellulose degradation mechanisms across the Tree of Life. - Current opinion in chemical biology
Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.Copyright Â© 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Identification and functional prediction of mitochondrial complex III and IV mutations associated with glioblastoma. - Neuro-oncology
Glioblastoma (GBM) is the most common primary brain tumor in adults, with a dismal prognosis. Treatment is hampered by GBM's unique biology, including differential cell response to therapy. Although several mitochondrial abnormalities have been identified, how mitochondrial DNA (mtDNA) mutations contribute to GBM biology and therapeutic response remains poorly described. We sought to determine the spectrum of functional complex III and IV mtDNA mutations in GBM.The complete mitochondrial genomes of 10 GBM cell lines were obtained using next-generation sequencing and combined with another set obtained from 32 GBM tissues. Three-dimensional structural mapping and analysis of all the nonsynonymous mutations identified in complex III and IV proteins was then performed to investigate functional importance.Over 200 mutations were identified in the mtDNAs, including a significant proportion with very low mutational loads. Twenty-five were nonsynonymous mutations in complex III and IV, 9 of which were predicted to be functional and affect mitochondrial respiratory chain activity. Most of the functional candidates were GBM specific and not found in the general population, and 2 were present in the germ-line. Patient-specific maps reveal that 43% of tumors carry at least one functional candidate.We reveal that the spectrum of GBM-associated mtDNA mutations is wider than previously thought, as well as novel structural-functional links between specific mtDNA mutations, abnormal mitochondria, and the biology of GBM. These results could provide tangible new prognostic indicators as well as targets with which to guide the development of patient-specific mitochondrially mediated chemotherapeutic approaches.Â© The Author(s) 2015. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.
Radiation damage to nucleoprotein complexes in macromolecular crystallography. - Journal of synchrotron radiation
Significant progress has been made in macromolecular crystallography over recent years in both the understanding and mitigation of X-ray induced radiation damage when collecting diffraction data from crystalline proteins. In contrast, despite the large field that is productively engaged in the study of radiation chemistry of nucleic acids, particularly of DNA, there are currently very few X-ray crystallographic studies on radiation damage mechanisms in nucleic acids. Quantitative comparison of damage to protein and DNA crystals separately is challenging, but many of the issues are circumvented by studying pre-formed biological nucleoprotein complexes where direct comparison of each component can be made under the same controlled conditions. Here a model protein-DNA complex C.Esp1396I is employed to investigate specific damage mechanisms for protein and DNA in a biologically relevant complex over a large dose range (2.07-44.63â€…MGy). In order to allow a quantitative analysis of radiation damage sites from a complex series of macromolecular diffraction data, a computational method has been developed that is generally applicable to the field. Typical specific damage was observed for both the protein on particular amino acids and for the DNA on, for example, the cleavage of base-sugar N1-C and sugar-phosphate C-O bonds. Strikingly the DNA component was determined to be far more resistant to specific damage than the protein for the investigated dose range. At low doses the protein was observed to be susceptible to radiation damage while the DNA was far more resistant, damage only being observed at significantly higher doses.
Structural and mutagenic analysis of the RM controller protein C.Esp1396I. - PloS one
Bacterial restriction-modification (RM) systems are comprised of two complementary enzymatic activities that prevent the establishment of foreign DNA in a bacterial cell: DNA methylation and DNA restriction. These two activities are tightly regulated to prevent over-methylation or auto-restriction. Many Type II RM systems employ a controller (C) protein as a transcriptional regulator for the endonuclease gene (and in some cases, the methyltransferase gene also). All high-resolution structures of C-protein/DNA-protein complexes solved to date relate to C.Esp1396I, from which the interactions of specific amino acid residues with DNA bases and/or the phosphate backbone could be observed. Here we present both structural and DNA binding data for a series of mutations to the key DNA binding residues of C.Esp1396I. Our results indicate that mutations to the backbone binding residues (Y37, S52) had a lesser affect on DNA binding affinity than mutations to those residues that bind directly to the bases (T36, R46), and the contributions of each side chain to the binding energies are compared. High-resolution X-ray crystal structures of the mutant and native proteins showed that the fold of the proteins was unaffected by the mutations, but also revealed variation in the flexible loop conformations associated with DNA sequence recognition. Since the tyrosine residue Y37 contributes to DNA bending in the native complex, we have solved the structure of the Y37F mutant protein/DNA complex by X-ray crystallography to allow us to directly compare the structure of the DNA in the mutant and native complexes.
Structural analysis of DNA-protein complexes regulating the restriction-modification system Esp1396I. - Acta crystallographica. Section F, Structural biology and crystallization communications
The controller protein of the type II restriction-modification (RM) system Esp1396I binds to three distinct DNA operator sequences upstream of the methyltransferase and endonuclease genes in order to regulate their expression. Previous biophysical and crystallographic studies have shown molecular details of how the controller protein binds to the operator sites with very different affinities. Here, two protein-DNA co-crystal structures containing portions of unbound DNA from native operator sites are reported. The DNA in both complexes shows significant distortion in the region between the conserved symmetric sequences, similar to that of a DNA duplex when bound by the controller protein (C-protein), indicating that the naked DNA has an intrinsic tendency to bend when not bound to the C-protein. Moreover, the width of the major groove of the DNA adjacent to a bound C-protein dimer is observed to be significantly increased, supporting the idea that this DNA distortion contributes to the substantial cooperativity found when a second C-protein dimer binds to the operator to form the tetrameric repression complex.
Structural analysis of mitochondrial mutations reveals a role for bigenomic protein interactions in human disease. - PloS one
Mitochondria are the energy producing organelles of the cell, and mutations within their genome can cause numerous and often severe human diseases. At the heart of every mitochondrion is a set of five large multi-protein machines collectively known as the mitochondrial respiratory chain (MRC). This cellular machinery is central to several processes important for maintaining homeostasis within cells, including the production of ATP. The MRC is unique due to the bigenomic origin of its interacting proteins, which are encoded in the nucleus and mitochondria. It is this, in combination with the sheer number of protein-protein interactions that occur both within and between the MRC complexes, which makes the prediction of function and pathological outcome from primary sequence mutation data extremely challenging. Here we demonstrate how 3D structural analysis can be employed to predict the functional importance of mutations in mtDNA protein-coding genes. We mined the MITOMAP database and, utilizing the latest structural data, classified mutation sites based on their location within the MRC complexes III and IV. Using this approach, four structural classes of mutation were identified, including one underexplored class that interferes with nuclear-mitochondrial protein interactions. We demonstrate that this class currently eludes existing predictive approaches that do not take into account the quaternary structural organization inherent within and between the MRC complexes. The systematic and detailed structural analysis of disease-associated mutations in the mitochondrial Complex III and IV genes significantly enhances the predictive power of existing approaches and our understanding of how such mutations contribute to various pathologies. Given the general lack of any successful therapeutic approaches for disorders of the MRC, these findings may inform the development of new diagnostic and prognostic biomarkers, as well as new drugs and targets for gene therapy.
Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance. - Proceedings of the National Academy of Sciences of the United States of America
Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.
A community continuity programme: volunteer faculty mentors and continuity learning. - The clinical teacher
Longitudinal generalist preceptorship experiences early in medical education can have beneficial effects on how students practise the art and science of medicine, regardless of their eventual career choices.We evaluated the first 2 years of implementation of an integrated, regional campus-based, early clinical experience programme, the Community Continuity Program, at our new community-based medical school that is under the supervision of volunteer primary care faculty members acting as continuity mentors (CMs). Curricular components for years 1 and 2 consisted of three annual 1-week community-based experiences with CMs, extensive physical diagnosis practice, interprofessional learning activities, a multigenerational family care experience, a mandatory Community Health Research Project (CHRP) in year 1 and a mandatory Quality Improvement Project in year 2.Outcome measures included student, faculty member and programme evaluations, student reflective narratives in portal-based e-journals, a Liaison Committee on Medical Education (LCME) self-study student survey and serial level-of-empathy surveys.â€‚ Students found all elements of this integrated community experience programme beneficial and worthwhile, especially the CMs and the use of standardised and real-life patients. CMs noted effective and professional student-patient interactions. The number of reflective e-journal postings per student during year1 ranged from 14 to 81 (mean, 47). Serial empathy questionnaires administered over 2 years demonstrated preservation of student empathy, and students believed that the programme had a positive effect on their personal level of empathy.â€‚ An integrative, longitudinal, community-based, early clinical experience programme driven by volunteer CMs provides patient-centered instruction for preclinical students in the clinical, social, behavioural, ethical and research foundations of medicine.Â© Blackwell Publishing Ltd 2013.
Non-rigid image registration to reduce beam-induced blurring of cryo-electron microscopy images. - Journal of synchrotron radiation
The typical dose used to record cryo-electron microscopy images from vitrified biological specimens is so high that radiation-induced structural alterations are bound to occur during data acquisition. Integration of all scattered electrons into one image can lead to significant blurring, particularly if the data are collected from an unsupported thin layer of ice suspended over the holes of a support film. Here, the dose has been fractioned and exposure series have been acquired in order to study beam-induced specimen movements under low dose conditions, prior to bubbling. Gold particles were added to the protein sample as fiducial markers. These were automatically localized and tracked throughout the exposure series and showed correlated motions within small patches, with larger amplitudes of motion vectors at the start of a series compared with the end of each series. A non-rigid scheme was used to register all images within each exposure series, using natural neighbor interpolation with the gold particles as anchor points. The procedure increases the contrast and resolution of the examined macromolecules.
Large multimeric assemblies of nucleosome assembly protein and histones revealed by small-angle X-ray scattering and electron microscopy. - The Journal of biological chemistry
The nucleosome assembly protein (NAP) family represents a key group of histone chaperones that are essential for cell viability. Several x-ray structures of NAP1 dimers are available; however, there are currently no structures of this ubiquitous chaperone in complex with histones. We have characterized NAP1 from Xenopus laevis and reveal that it forms discrete multimers with histones H2A/H2B and H3/H4 at a stoichiometry of one NAP dimer to one histone fold dimer. These complexes have been characterized by size exclusion chromatography, analytical ultracentrifugation, multiangle laser light scattering, and small-angle x-ray scattering to reveal their oligomeric assembly states in solution. By employing single-particle cryo-electron microscopy, we visualized these complexes for the first time and show that they form heterogeneous ring-like structures, potentially acting as large scaffolds for histone assembly and exchange.
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