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Biophysics
Atomic force microscopy for single molecule characterisation of protein aggregation.
Feb 11, 2019   Archives Of Biochemistry And Biophysics
Ruggeri FS, Šneideris T, Vendruscolo M, Knowles TPJ
Atomic force microscopy for single molecule characterisation of protein aggregation.
Feb 11, 2019
Archives Of Biochemistry And Biophysics
The development of atomic force microscopy (AFM) has opened up a wide range of novel opportunities in nanoscience and new modalities of observation in complex biological systems. AFM imaging has been widely employed to resolve the complex and heterogeneous conformational states involved in protein aggregation at the single molecule scale and shed light onto the molecular basis of a variety of human pathologies, including neurodegenerative disorders. The study of individual macromolecules at nanoscale, however, remains challenging, especially when fully quantitative information is required. In this review, we first discuss the principles of AFM with a special emphasis on the fundamental factors defining its sensitivity and accuracy. We then review the fundamental parameters and approaches to work at the limit of AFM resolution in order to perform single molecule statistical analysis of biomolecules and nanoscale protein aggregates. This single molecule statistical approach has proved to be powerful to unravel the molecular and hierarchical assembly of the misfolded species present transiently during protein aggregation, to visualise their dynamics at the nanoscale, as well to study the structural properties of amyloid-inspired functional nanomaterials.
Altered human oligodendrocyte heterogeneity in multiple sclerosis.
Feb 13, 2019   Nature Add nature.com free-link Cancel
Jäkel S, Agirre E, Falcão AM, van Bruggen D, Lee KW, Knuesel I, Malhotra D, Ffrench-Constant C, Williams A, Castelo-Branco G
Altered human oligodendrocyte heterogeneity in multiple sclerosis.
Feb 13, 2019
Nature
Oligodendrocyte (OL) pathology is increasingly implicated in neurodegenerative diseases as OLs both myelinate and provide metabolic support to axons. In multiple sclerosis (MS), demyelination in the central nervous system (CNS) thus leads to neurodegeneration, but the severity of MS between patients is very variable. Disability does not correlate well with the extent of demyelination1, suggesting that other factors contribute to this variability. One such factor may be OL heterogeneity. Not all OLs are the same-mouse spinal cord OLs inherently produce longer myelin sheaths than cortical OLs2, and single-cell analysis of mouse CNS identified further differences3,4. However, the extent of human OL heterogeneity and its possible contribution to MS pathology remains unknown. Here we performed single nuclei RNA-sequencing (snRNA-seq) from white matter (WM) areas of post mortem human brain both in control (Ctr) and MS patients. We identified sub-clusters of oligodendroglia in Ctr human WM, some similar to mouse, and defined new markers for these cell states. Strikingly, some sub-clusters were under-represented in MS tissue, while others were more prevalent. These differences in mature OL sub-clusters may indicate different functional states of OLs in MS lesions. Since this is similar in normal appearing white matter (NAWM), MS is a more diffuse disease than its focal demyelination suggests. Our findings of an altered oligodendroglial heterogeneity in MS may be important to understanding disease progression and developing therapeutic approaches.
The histone demethylase PHF8 facilitates alternative splicing of the histocompatibility antigen HLA-G.
Feb 13, 2019   FEBS Letters
Leisegang MS, Gu L, Preussner J, Günther S, Hitzel J, Ratiu C, Weigert A, Chen W, Schwarz EC, Looso M, Fork C, Brandes RP
The histone demethylase PHF8 facilitates alternative splicing of the histocompatibility antigen HLA-G.
Feb 13, 2019
FEBS Letters
Histone3-Lysine9 (H3K9) residues not only control gene expression, but also contribute to RNA splicing. Here, the H3K9 histone demethylase PHF8 was investigated in endothelial cells for its involvement in alternative splicing. An angiogenic sprouting assay shows the importance of PHF8 for endothelial cells. Immunoprecipitation reveals that PHF8 interacts with U1 spliceosomal proteins, such as SRPK1 and snRNP70. We identify the histocompatibility antigen HLA-G as a target of PHF8. The inclusion of HLA-G intron-4, with concomitant RNA Polymerase II accumulation at this intron, is controlled by PHF8 and H3K9. Soluble-HLA-G is generated after PHF8 knockdown, which leads to reduced T cell proliferation. Collectively, PHF8 knockdown generates the immunosuppressive alternative splice product soluble-HLA-G, which is secreted by endothelial cells to elicit a potential inhibitory effect on inflammation. This article is protected by copyright. All rights reserved.
A biophysical model explains the spontaneous bursting behavior in the developing retina.
Feb 13, 2019   Scientific Reports
Matzakos-Karvouniari D, Gil L, Orendorff E, Marre O, Picaud S, Cessac B
A biophysical model explains the spontaneous bursting behavior in the developing retina.
Feb 13, 2019
Scientific Reports
During early development, waves of activity propagate across the retina and play a key role in the proper wiring of the early visual system. During a particular phase of the retina development (stage II) these waves are triggered by a transient network of neurons, called Starburst Amacrine Cells (SACs), showing a bursting activity which disappears upon further maturation. The underlying mechanisms of the spontaneous bursting and the transient excitability of immature SACs are not completely clear yet. While several models have attempted to reproduce retinal waves, none of them is able to mimic the rhythmic autonomous bursting of individual SACs and reveal how these cells change their intrinsic properties during development. Here, we introduce a mathematical model, grounded on biophysics, which enables us to reproduce the bursting activity of SACs and to propose a plausible, generic and robust, mechanism that generates it. The core parameters controlling repetitive firing are fast depolarizing V-gated calcium channels and hyperpolarizing V-gated potassium channels. The quiescent phase of bursting is controlled by a slow after hyperpolarization (sAHP), mediated by calcium-dependent potassium channels. Based on a bifurcation analysis we show how biophysical parameters, regulating calcium and potassium activity, control the spontaneously occurring fast oscillatory activity followed by long refractory periods in individual SACs. We make a testable experimental prediction on the role of voltage-dependent potassium channels on the excitability properties of SACs and on the evolution of this excitability along development. We also propose an explanation on how SACs can exhibit a large variability in their bursting periods, as observed experimentally within a SACs network as well as across different species, yet based on a simple, unique, mechanism. As we discuss, these observations at the cellular level have a deep impact on the retinal waves description.
Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins.
Feb 13, 2019   Scientific Reports
Buhrke D, Tavraz NN, Shcherbakova DM, Sauthof L, Moldenhauer M, Vélazquez Escobar F, Verkhusha VV, Hildebrandt P, Friedrich T
Chromophore binding to two cysteines increases quantum yield of near-infrared fluorescent proteins.
Feb 13, 2019
Scientific Reports
Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness. Here, by combining biochemical, spectroscopic and resonance Raman (RR) assays, we purified and characterized an iRFP variant that contains a BV chromophore simultaneously bound to two cysteines. This protein with the unusual double-Cys attached BV showed the highest fluorescence quantum yield (FQY) of 16.6% reported for NIR FPs, whereas the initial iRFP appeared to be a mixture of species with a mean FQY of 11.1%. The purified protein was also characterized with 1.3-fold higher extinction coefficient that together with FQY resulted in almost two-fold brighter fluorescence than the original iRFP as isolated. This work shows that the high FQY of iRFPs with two cysteines is a direct consequence of the double attachment. The PAS-Cys, GAF-Cys and double-Cys attachment each entails distinct configurational constraints of the BV adduct, which can be identified by distinct RR spectroscopic features, i.e. the marker band including the C=C stretching coordinate of the ring A-B methine bridge, which was previously identified as being characteristic for rigid chromophore embedment and high FQY. Our findings can be used to rationally engineer iRFP variants with enhanced FQYs.
Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels.
Feb 13, 2019   Scientific Reports
Ko J, Myeong J, Shin YC, So I
Differential PI(4,5)P2 sensitivities of TRPC4, C5 homomeric and TRPC1/4, C1/5 heteromeric channels.
Feb 13, 2019
Scientific Reports
Transient receptor potential canonical (TRPC) 4 and TRPC5 channels are modulated by the Gαq-PLC pathway. Since phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) maintains TRPC4 and TRPC5 channel function, the Gαq-PLC pathway inhibits channel activity by depleting PI(4,5)P2. Here we investigated the difference in PI(4,5)P2 sensitivity between homomeric and heteromeric TRPC channels. First, by using a Danio rerio voltage-sensing phosphatase (DrVSP), we show that PI(4,5)P2 dephosphorylation robustly inhibits TRPC4α, TRPC4β, and TRPC5 homotetramer currents and also TRPC1/4α, TRPC1/4β, and TRPC1/5 heterotetramer currents. Secondly, sensitivity of channels to PI(4,5)P2 dephosphorylation was suggested through the usage of FRET in combination with patch clamping. The sensitivity increased in the sequence TRPC4β < TRPC4α < TRPC5 in homotetramers, whereas when forming heterotetramers with TRPC1, the sensitivity was approximately equal between the channels. Thirdly, we determined putative PI(4,5)P2 binding sites based on a TRPC4 prediction model. By neutralization of basic residues, we identified putative PI(4,5)P2 binding sites because the mutations reduced FRET to a PI(4,5)P2 sensor and reduced the current amplitude. Therefore, one functional TRPC4 has 8 pockets with the two main binding regions; K419, K664/R511, K518, H630. We conclude that TRPC1 channel function as a regulator in setting PI(4,5)P2 affinity for TRPC4 and TRPC5 that changes PI(4,5)P2 sensitivity.
Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.
Feb 13, 2019   Proceedings Of The National Academy Of Sciences Of The United States Of America
Campbell NG, Shekar A, Aguilar JI, Peng D, Navratna V,   . . . . . .   , Bellan LM, Matthies HJG, Gouaux E, Mchaourab HS, Galli A
Structural, functional, and behavioral insights of dopamine dysfunction revealed by a deletion in SLC6A3.
Feb 13, 2019
Proceedings Of The National Academy Of Sciences Of The United States Of America
The human dopamine (DA) transporter (hDAT) mediates clearance of DA. Genetic variants in hDAT have been associated with DA dysfunction, a complication associated with several brain disorders, including autism spectrum disorder (ASD). Here, we investigated the structural and behavioral bases of an ASD-associated in-frame deletion in hDAT at N336 (∆N336). We uncovered that the deletion promoted a previously unobserved conformation of the intracellular gate of the transporter, likely representing the rate-limiting step of the transport process. It is defined by a "half-open and inward-facing" state (HOIF) of the intracellular gate that is stabilized by a network of interactions conserved phylogenetically, as we demonstrated in hDAT by Rosetta molecular modeling and fine-grained simulations, as well as in its bacterial homolog leucine transporter by electron paramagnetic resonance analysis and X-ray crystallography. The stabilization of the HOIF state is associated both with DA dysfunctions demonstrated in isolated brains of Drosophila melanogaster expressing hDAT ∆N336 and with abnormal behaviors observed at high-time resolution. These flies display increased fear, impaired social interactions, and locomotion traits we associate with DA dysfunction and the HOIF state. Together, our results describe how a genetic variation causes DA dysfunction and abnormal behaviors by stabilizing a HOIF state of the transporter.
The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2, a phosphatidylserine flippase.
Feb 13, 2019   The Biochemical Journal
Theorin L, Faxen K, Sørensen DM, Migotti R, Dittmar G, Schiller J, Daleke DL, Palmgren M, López-Marqués RL, Günther Pomorski T
The lipid head group is the key element for substrate recognition by the P4 ATPase ALA2, a phosphatidylserine flippase.
Feb 13, 2019
The Biochemical Journal
Type IV P-type ATPases (P4 ATPases) are lipid flippases that catalyze phospholipid transport from the exoplasmic to the cytoplasmic leaflet of cellular membranes, but the mechanism by which they recognize and transport phospholipids through the lipid bilayer remains unknown. In this study, we succeeded in purifying recombinant Aminophospholipid ATPase 2 (ALA2), a member of the P4 ATPase subfamily in Arabidopsis thaliana , in complex with the ALA-Interacting Subunit 5 (ALIS5). The ATP hydrolytic activity of the ALA2-ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Small changes in the stereochemistry or the functional groups of the phosphatidylserine head group affected enzymatic activity, whereas alteration in the length and composition of the acyl chains only had minor effects. Likewise enzymatic activity of the ALA2-ALIS5 complex was stimulated by both mono- and di-acyl phosphatidylserines. Taken together, the results identify the lipid head group as the key structural element for substrate recognition by the P4 ATPase.
Proinflammatory and Amyloidogenic S100A9 Induced by Traumatic Brain Injury in Mouse Model.
Feb 12, 2019   Neuroscience Letters
Wang C, Iashchishyn IA, Kara J, Foderà V, Vetri V, Sancataldo G, Marklund N, Morozova-Roche LA
Proinflammatory and Amyloidogenic S100A9 Induced by Traumatic Brain Injury in Mouse Model.
Feb 12, 2019
Neuroscience Letters
Traumatic brain injury (TBI) represents a significant risk factor for development of neurodegenerative diseases such as Alzheimer's and Parkinson's. The S100A9-driven amyloid-neuroinflammatory cascade occurring during primary and secondary TBI events can serve as a mechanistic link between TBI and Alzheimer's as demonstrated recently in the human brain tissues. Here by using immunohistochemistry in the controlled cortical impact TBI mouse model we have found pro-inflammatory S100A9 in the brain tissues of all mice on the first and third post-TBI days, while 70% of mice did not show any S100A9 presence on seventh post-TBI day similar to controls. This indicates that defensive mechanisms effectively cleared S100A9 in these mouse brain tissues during post-TBI recovery. By using sequential immunohistochemistry we have shown that S100A9 was produced by both neuronal and microglial cells. However, Aβ peptide deposits characteristic for Alzheimer's disease were not detected in any post-TBI animals. On the first and third post-TBI days S100A9 was found to aggregate intracellularly into amyloid oligomers, similar to what was previously observed in human TBI tissues. Complementary, by using Rayleigh scatting, intrinsic fluorescence and atomic force microscopy we demonstrated that in vitro S100A9 self-assembles into amyloid oligomers within minutes. Its amyloid aggregation is highly dependent on changes of environmental conditions such as variation of calcium levels, pH, temperature and reduction/oxidation, which might be relevant to perturbation of cellular and tissues homeostasis under TBI. Present results demonstrate that S100A9 induction mechanisms in TBI are similar in mice and humans, emphasizing that S100A9 is an important marker of brain injury and therefore can be a potential therapeutic target.
Analyses of association of psoriatic arthritis and psoriasis vulgaris with functional NCF1 variants.
Feb 12, 2019   Rheumatology (Oxford, England)
Löhr S, Ekici AB, Uebe S, Büttner C, Köhm M,   . . . . . .   , Rech J, Holmdahl R, Burkhardt H, Reis A, Hüffmeier U
Persistent upregulation of the β-tubulin tubb6, linked to muscle regeneration, is a source of microtubule disorganization in dystrophic muscle.
Feb 12, 2019   Human Molecular Genetics
Randazzo D, Khalique U, Belanto JJ, Kenea A, Talsness DM, Olthoff JT, Tran MD, Zaal KJ, Pak K, Pinal-Fernandez I, Mammen AL, Sackett D, Ervasti JM, Ralston E
Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.
Feb 12, 2019   The Plant Journal : For Cell And Molecular Biology
Skalák J, Vercruyssen L, Claeys H, Hradilová J, Černý M,   . . . . . .   , Dhondt S, Koukalová Š, Zouhar J, Inzé D, Brzobohatý B
Multifaceted activity of cytokinin in leaf development shapes its size and structure in Arabidopsis.
Feb 12, 2019
The Plant Journal : For Cell And Molecular Biology
Phytohormone cytokinin has been shown to affect many aspects of plant development ranging from the regulation of the shoot apical meristem to leaf senescence. However some studies reported contradictory effects of cytokinin on leaf physiology. Thus, cytokinin treatments cause both chlorosis and increased greening, and both decrease and increase in cell size. To elucidate this multifaceted role of cytokinin in leaf development, we have employed a system of temporal control over the cytokinin pool and investigated the consequences of modulated cytokinin levels in the third leaf of Arabidopsis. We show that at the cell proliferation phase, cytokinin is needed to maintain cell proliferation by blocking the transition to cell expansion and the onset of photosynthesis. Transcriptome profiling revealed regulation by cytokinin of a gene suit previously shown to affect cell proliferation and expansion, and thereby a molecular mechanism by which cytokinin modulates a molecular network underlying the cellular responses. During the cell expansion phase, cytokinin stimulates cell expansion and differentiation. Consequently, a cytokinin excess at the cell expansion phase results in an increased leaf and rosette size fueled by higher cell expansion rate, yielding higher shoot biomass. Proteome profiling revealed the stimulation of primary metabolism by cytokinin, in line with an increased sugar content that is expected to increase turgor pressure, representing the driving force of cell expansion. Thus, the developmental timing of cytokinin content fluctuations, together with a tight control of primary metabolism, is a key factor mediating transitions from cell proliferation to cell expansion in leaves. This article is protected by copyright. All rights reserved.
Effects of long-term exposure to elevated temperature on Zea mays endosperm development during grain fill.
Feb 12, 2019   The Plant Journal : For Cell And Molecular Biology
Boehlein SK, Liu P, Webster A, Ribeiro C, Suzuki M,   . . . . . .   , McCarty DR, Koch KE, Hannah LC, Hennen-Bierwagen TA, Myers AM
Effects of long-term exposure to elevated temperature on Zea mays endosperm development during grain fill.
Feb 12, 2019
The Plant Journal : For Cell And Molecular Biology
Cereal yields decrease when grain-fill proceeds under conditions of prolonged, moderately-elevated temperatures. Endosperm-endogenous processes alter both rate and duration of dry-weight gain, but underlying mechanisms remain unclear. Heat effects could be mediated by either abnormal, premature cessation of storage compound deposition or accelerated implementation of normal development. This study used controlled environments to isolate temperature as the sole environmental variable during Zea mays kernel-fill, from 12 days after pollination to maturity. Plants subjected to elevated day, elevated night temperatures (38°C day, 28°C night [38/28°C]) or elevated day, normal night (38/17°C), were compared to those from controls grown in normal day and night conditions (28/17°C). Progression of change over time in endosperm tissue was followed to dissect contributions at multiple levels, including transcriptome, metabolome, enzyme activities, product accumulation, and tissue ultrastructure. Integrated analyses indicated that the normal developmental program of endosperm is fully executed under prolonged high-temperature conditions, but at a faster rate. Accelerated development was observed when both day and night temperatures were elevated, but not whan daytime temperature alone was increased. Although transcripts for most components of glycolysis and respiration were either up-regulated or minimally affected, elevated temperatures decreased abundance of mRNAs related to biosynthesis of starch and storage proteins. Further analysis of 20 central-metabolic enzymes revealed six activities that were reduced under high-temperature conditions, indicating candidate roles in the observed reduction of grain dry weight. Nonetheless, a striking overall resilience of grain-filling in the face of elevated temperatures can be attributed to acceleration of normal endosperm development. This article is protected by copyright. All rights reserved.
Evaluation of novel imaging devices for nanoparticle-mediated fluorescence-guided lung tumor therapy.
Feb 11, 2019   The Annals Of Thoracic Surgery
Kinoshita T, Ujiie H, Chen J, Ding L, Chan H,   . . . . . .   , Weersink R, Wilson BC, Zheng G, Asamura H, Yasufuku K
Evaluation of novel imaging devices for nanoparticle-mediated fluorescence-guided lung tumor therapy.
Feb 11, 2019
The Annals Of Thoracic Surgery
BACKGROUND: Non-surgical and minimally-invasive approaches for early stage peripheral lung cancer are needed to avoid the known morbidity of surgery, particularly in high-risk patients. We have previously demonstrated the utility of multifunctional porphyrin-phospholipid nanoparticles (porphysomes) for fluorescence imaging and phototherapy following preferential accumulation into tumors. The objective of this study was to demonstrate the feasibility of porphysome-mediated imaging and photothermal therapy using a newly-developed fiberscope and thoracoscope. METHODS: To prepare this technology for clinical translation, we developed a porphysome-specific fiberscope (scanning fiber endoscope [SFE]) and porphysome-specific thoracoscope (P-PINPOINT), both capable of detecting porphysome fluorescence, for image-guided transbronchial and transpleural photothermal therapy to treat endo/peribronchial and subpleural tumors, respectively. These were tested in three animal models: human lung cancer xenografts (A549) in mice, orthotopic VX2 lung tumors in rabbits, and ex vivo pig lung into which A549 tumor tissue was transplanted. RESULTS: The SFE, whose 1.2 mm diameter is small enough to pass through the working channel of a conventional bronchoscope, could visualize porphysome-laden tumors located inside or close to the peripheral bronchial wall. The P-PINPOINT system had high sensitivity for porphysome fluorescence and enabled image-guided thoracoscopic resection of porphysome-accumulating tumors close to the pleura. Porphysomes also enhanced the efficacy of SFE-guided transbronchial photothermal therapy and P-PINPOINT-guided transpleural photothermal therapy, resulting in selective and efficient tumor tissue ablation in the rabbit and pig models. CONCLUSIONS: These results support the potential for clinical translation of this novel platform to impact non-surgical and minimally-invasive treatment options for early stage peripheral lung cancer.
Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors.
Feb 16, 2019   Science Advances
Dominy SS, Lynch C, Ermini F, Benedyk M, Marczyk A,   . . . . . .   , Reynolds EC, Faull RLM, Curtis MA, Dragunow M, Potempa J
Porphyromonas gingivalis in Alzheimer's disease brains: Evidence for disease causation and treatment with small-molecule inhibitors.
Feb 16, 2019
Science Advances
Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, was identified in the brain of Alzheimer's disease patients. Toxic proteases from the bacterium called gingipains were also identified in the brain of Alzheimer's patients, and levels correlated with tau and ubiquitin pathology. Oral P. gingivalis infection in mice resulted in brain colonization and increased production of Aβ1-42, a component of amyloid plaques. Further, gingipains were neurotoxic in vivo and in vitro, exerting detrimental effects on tau, a protein needed for normal neuronal function. To block this neurotoxicity, we designed and synthesized small-molecule inhibitors targeting gingipains. Gingipain inhibition reduced the bacterial load of an established P. gingivalis brain infection, blocked Aβ1-42 production, reduced neuroinflammation, and rescued neurons in the hippocampus. These data suggest that gingipain inhibitors could be valuable for treating P. gingivalis brain colonization and neurodegeneration in Alzheimer's disease.
Cell scientists to watch - Franziska Lautenschläger.
Feb 12, 2019   Journal Of Cell Science
Cell scientists to watch - Franziska Lautenschläger.
Feb 12, 2019
Journal Of Cell Science
 
Franziska Lautenschläger studied physics at the University of Leipzig, Germany and Université Paul Sabatier Toulouse, France, before graduating with a physics diploma from Leipzig. For her PhD on mechanical changes during stem cell differentiation, she joined the laboratory of Jochen Guck at the University of Cambridge, UK. In 2011, Franziska moved to Paris, France, for her postdoctoral work on the migration of immune cells under confinement at the Institut Curie with supervisor Matthieu Piel. Since 2013, she has been an independent group leader and holds a junior professorship in biophysics from Saarland University, Saarbrücken, Germany. In addition, the Leibniz Institute for New Materials, Saarbrücken, appointed her as junior group leader in 2017. Franziska's research focuses on cell migration and polarity, and the links to cell mechanics and the different cytoskeletal networks in cells.
Spiraling in Control: Structures and Mechanisms of the Hsp104 Disaggregase.
Feb 12, 2019   Cold Spring Harbor Perspectives In Biology
Shorter J, Southworth DR
Spiraling in Control: Structures and Mechanisms of the Hsp104 Disaggregase.
Feb 12, 2019
Cold Spring Harbor Perspectives In Biology
Hsp104 is a hexameric AAA+ ATPase and protein disaggregase found in yeast, which couples ATP hydrolysis to the dissolution of diverse polypeptides trapped in toxic preamyloid oligomers, phase-transitioned gels, disordered aggregates, amyloids, and prions. Hsp104 shows plasticity in disaggregating diverse substrates, but how its hexameric architecture operates as a molecular machine has remained unclear. Here, we highlight structural advances made via cryoelectron microscopy (cryo-EM) that enhance our mechanistic understanding of Hsp104 and other related AAA+ translocases. Hsp104 hexamers are dynamic and adopt open "lock-washer" spiral states and closed ring structures that envelope polypeptide substrate inside the axial channel. ATP hydrolysis-driven conformational changes at the spiral seam ratchet substrate deeper into the channel. Remarkably, this mode of polypeptide translocation is reminiscent of models for how hexameric helicases unwind DNA and RNA duplexes. Thus, Hsp104 likely adapts elements of a deeply rooted, ring-translocase mechanism to the specialized task of protein disaggregation.
Gliotoxin from Aspergillus fumigatus Abrogates Leukotriene B4 Formation through Inhibition of Leukotriene A4 Hydrolase.
Feb 12, 2019   Cell Chemical Biology
König S, Pace S, Pein H, Heinekamp T, Kramer J,   . . . . . .   , Hertweck C, Brakhage AA, Gerstmeier J, Proschak E, Werz O
Gliotoxin from Aspergillus fumigatus Abrogates Leukotriene B4 Formation through Inhibition of Leukotriene A4 Hydrolase.
Feb 12, 2019
Cell Chemical Biology
The epidithiodioxopiperazine gliotoxin is a virulence factor of Aspergillus fumigatus, the most important airborne fungal pathogen of humans. Gliotoxin suppresses innate immunity in invasive aspergillosis, particularly by compromising neutrophils, but the underlying molecular mechanisms remain elusive. Neutrophils are the first responders among innate immune cells recruited to sites of infection by the chemoattractant leukotriene (LT)B4 that is biosynthesized by 5-lipoxygenase and LTA4 hydrolase (LTA4H). Here, we identified gliotoxin as inhibitor of LTA4H that selectively abrogates LTB4 formation in human leukocytes and in distinct animal models. Gliotoxin failed to inhibit the formation of other eicosanoids and the aminopeptidase activity of the bifunctional LTA4H. Suppression of LTB4 formation by gliotoxin required the cellular environment and/or reducing conditions, and only the reduced form of gliotoxin inhibited LTA4H activity. Conclusively, gliotoxin suppresses the biosynthesis of the potent neutrophil chemoattractant LTB4 by direct interference with LTA4H thereby impairing neutrophil functions in invasive aspergillosis.
De Novo Structural Pattern Mining in Cellular Electron Cryotomograms.
Feb 12, 2019   Structure (London, England : 1993)
Xu M, Singla J, Tocheva EI, Chang YW, Stevens RC, Jensen GJ, Alber F
De Novo Structural Pattern Mining in Cellular Electron Cryotomograms.
Feb 12, 2019
Structure (London, England : 1993)
Electron cryotomography enables 3D visualization of cells in a near-native state at molecular resolution. The produced cellular tomograms contain detailed information about a plethora of macromolecular complexes, their structures, abundances, and specific spatial locations in the cell. However, extracting this information in a systematic way is very challenging, and current methods usually rely on individual templates of known structures. Here, we propose a framework called "Multi-Pattern Pursuit" for de novo discovery of different complexes from highly heterogeneous sets of particles extracted from entire cellular tomograms without using information of known structures. These initially detected structures can then serve as input for more targeted refinement efforts. Our tests on simulated and experimental tomograms show that our automated method is a promising tool for supporting large-scale template-free visual proteomics analysis.
Allostery in Its Many Disguises: From Theory to Applications.
Feb 12, 2019   Structure (London, England : 1993)
Wodak SJ, Paci E, Dokholyan NV, Berezovsky IN, Horovitz A,   . . . . . .   , Tsai CJ, Jang H, Padhorny D, Kozakov D, McLeish T
Allostery in Its Many Disguises: From Theory to Applications.
Feb 12, 2019
Structure (London, England : 1993)
Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used here to provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.
Microtubule End-Clustering Maintains a Steady-State Spindle Shape.
Feb 12, 2019   Current Biology : CB
Hueschen CL, Galstyan V, Amouzgar M, Phillips R, Dumont S
Microtubule End-Clustering Maintains a Steady-State Spindle Shape.
Feb 12, 2019
Current Biology : CB
Each time a cell divides, the microtubule cytoskeleton self-organizes into the metaphase spindle: an ellipsoidal steady-state structure that holds its stereotyped geometry despite microtubule turnover and internal stresses [1-6]. Regulation of microtubule dynamics, motor proteins, microtubule crosslinking, and chromatid cohesion can modulate spindle size and shape, and yet modulated spindles reach and hold a new steady state [7-11]. Here, we ask what maintains any spindle steady-state geometry. We report that clustering of microtubule ends by dynein and NuMA is essential for mammalian spindles to hold a steady-state shape. After dynein or NuMA deletion, the mitotic microtubule network is "turbulent"; microtubule bundles extend and bend against the cell cortex, constantly remodeling network shape. We find that spindle turbulence is driven by the homotetrameric kinesin-5 Eg5, and that acute Eg5 inhibition in turbulent spindles recovers spindle geometry and stability. Inspired by in vitro work on active turbulent gels of microtubules and kinesin [12, 13], we explore the kinematics of this in vivo turbulent network. We find that turbulent spindles display decreased nematic order and that motile asters distort the nematic director field. Finally, we see that turbulent spindles can drive both flow of cytoplasmic organelles and whole-cell movement-analogous to the autonomous motility displayed by droplet-encapsulated turbulent gels [12]. Thus, end-clustering by dynein and NuMA is required for mammalian spindles to reach a steady-state geometry, and in their absence Eg5 powers a turbulent microtubule network inside mitotic cells.
Dual Action of the PN159/KLAL/MAP Peptide: Increase of Drug Penetration across Caco-2 Intestinal Barrier Model by Modulation of Tight Junctions and Plasma Membrane Permeability.
Feb 12, 2019   Pharmaceutics
Bocsik A, Gróf I, Kiss L, Ötvös F, Zsíros O, Daruka L, Fülöp L, Vastag M, Kittel Á, Imre N, Martinek TA, Pál C, Szabó-Révész P, Deli MA
Dual Action of the PN159/KLAL/MAP Peptide: Increase of Drug Penetration across Caco-2 Intestinal Barrier Model by Modulation of Tight Junctions and Plasma Membrane Permeability.
Feb 12, 2019
Pharmaceutics
The absorption of drugs is limited by the epithelial barriers of the gastrointestinal tract. One of the strategies to improve drug delivery is the modulation of barrier function by the targeted opening of epithelial tight junctions. In our previous study the 18-mer amphiphilic PN159 peptide was found to be an effective tight junction modulator on intestinal epithelial and blood⁻brain barrier models. PN159, also known as KLAL or MAP, was described to interact with biological membranes as a cell-penetrating peptide. In the present work we demonstrated that the PN159 peptide as a penetration enhancer has a dual action on intestinal epithelial cells. The peptide safely and reversibly enhanced the permeability of Caco-2 monolayers by opening the intercellular junctions. The penetration of dextran molecules with different size and four efflux pump substrate drugs was increased several folds. We identified claudin-4 and -7 junctional proteins by docking studies as potential binding partners and targets of PN159 in the opening of the paracellular pathway. In addition to the tight junction modulator action, the peptide showed cell membrane permeabilizing and antimicrobial effects. This dual action is not general for cell-penetrating peptides (CPPs), since the other three CPPs tested did not show barrier opening effects.
4-Cyanoindole-2'-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics.
Feb 12, 2019   Molecules (Basel, Switzerland)
Ahmed IA, Acharyya A, Eng CM, Rodgers JM, DeGrado WF, Jo H, Gai F
4-Cyanoindole-2'-deoxyribonucleoside as a Dual Fluorescence and Infrared Probe of DNA Structure and Dynamics.
Feb 12, 2019
Molecules (Basel, Switzerland)
Unnatural nucleosides possessing unique spectroscopic properties that mimic natural nucleobases in both size and chemical structure are ideally suited for spectroscopic measurements of DNA/RNA structure and dynamics in a site-specific manner. However, such unnatural nucleosides are scarce, which prompts us to explore the utility of a recently found unnatural nucleoside, 4-cyanoindole-2'-deoxyribonucleoside (4CNI-NS), as a site-specific spectroscopic probe of DNA. A recent study revealed that 4CNI-NS is a universal nucleobase that maintains the high fluorescence quantum yield of 4-cyanoindole and that among the four natural nucleobases, only guanine can significantly quench its fluorescence. Herein, we further show that the C≡N stretching frequency of 4CNI-NS is sensitive to the local environment, making it a useful site-specific infrared probe of oligonucleotides. In addition, we demonstrate that the fluorescence-quencher pair formed by 4CNI-NS and guanine can be used to quantitatively assess the binding affinity of a single-stranded DNA to the protein system of interest via fluorescence spectroscopy, among other applications. We believe that this fluorescence binding assay is especially useful as its potentiality allows high-throughput screening of DNA⁻protein interactions.
Role of gonadotropin-releasing hormone II in the mammalian nervous system.
Feb 12, 2019   Expert Review Of Endocrinology & Metabolism
Kauffman AS, Rissman EF
Role of gonadotropin-releasing hormone II in the mammalian nervous system.
Feb 12, 2019
Expert Review Of Endocrinology & Metabolism
Gonadotropin-releasing hormone (GnRH) is a small neuropeptide of which there are multiple structural variants. The first variant identified in mammals, GnRH I, controls the release of pituitary gonadotropins. More recently, a second isoform, GnRH II, first isolated in the bird, was identified in the mammalian brain and periphery. Although it is unlikely to be a primary regulator of gonadotropin release, GnRH II appears to have a wide array of physiological and behavioral functions. GnRH II-containing fibers are present in several nuclei known to regulate reproduction and/or feeding, and its concentration in several of these areas fluctuates in response to changes in food availability, and thus energetic status. In musk shrews, GnRH II acts as a permissive regulator of female reproductive behavior based on energy status, as well as an inhibitor of short-term food intake. In this regard, GnRH II is similar to leptin, neuropeptide Y and several other neurotransmitters that regulate both feeding and reproduction. At least two GnRH receptors are present in the mammalian brain, and increasing evidence suggests that the behavioral effects of GnRH II are mediated by receptor subtypes distinct from the type-1 GnRH receptor (which mediates GnRH I action); the most probable candidate is the type-2 GnRH receptor. GnRH II also regulates the density and/or activity of calcium and potassium channels in the nervous systems of amphibians and fish, a function that may also exist in mammalian neurons. It is likely that the highly conserved GnRH II system has been co-opted over evolutionary time to possess multiple regulatory functions in a broad range of neurobiological aspects.
Architectural principles for Hfq/Crc-mediated regulation of gene expression.
Feb 13, 2019   ELife
Pei XY, Dendooven T, Sonnleitner E, Chen S, Blasi U, Luisi BF
Architectural principles for Hfq/Crc-mediated regulation of gene expression.
Feb 13, 2019
ELife
In diverse bacterial species, the global regulator Hfq contributes to post-transcriptional networks that control expression of numerous genes. Hfq of the opportunistic pathogen Pseudomonas aeruginosa inhibits translation of target transcripts by forming a regulatory complex with the catabolite repression protein Crc. This repressive complex acts part of an intricate mechanism of preferred nutrient utilisation. We describe high-resolution cryo-EM structures of the assembly of Hfq and Crc bound to the translation initiation site of a target mRNA. The core of the assembly is formed through interactions of two cognate RNAs, two Hfq hexamers and a Crc pair. Additional Crc protomers are recruited to the core to generate higher-order assemblies with demonstrated regulatory activity in vivo. This study reveals how Hfq cooperates with a partner protein to regulate translation, and provides a structural basis for an RNA code that guides global regulators to interact cooperatively and regulate different RNA targets.

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