scholarly journals Phosphorylation of the Bruchpilot N-terminus in Drosophila unlocks axonal transport of active zone building blocks

2019 ◽  
Vol 132 (6) ◽  
pp. jcs225151 ◽  
Author(s):  
Jan H. Driller ◽  
Janine Lützkendorf ◽  
Harald Depner ◽  
Matthias Siebert ◽  
Benno Kuropka ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Active Zone ◽  
Building Blocks ◽  
N Terminus

scholarly journals Maturation of active zone assembly by Drosophila Bruchpilot

2009 ◽  
Vol 186 (1) ◽  
pp. 129-145 ◽  
Author(s):  
Wernher Fouquet ◽  
David Owald ◽  
Carolin Wichmann ◽  
Sara Mertel ◽  
Harald Depner ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Drosophila Melanogaster ◽  
High Resolution ◽  
Active Zone ◽  
Synaptic Vesicles ◽  
C Terminus ◽  
Family Protein ◽  
N Terminus ◽  
Dense Projections ◽  
Ca2 Channels

Synaptic vesicles fuse at active zone (AZ) membranes where Ca2+ channels are clustered and that are typically decorated by electron-dense projections. Recently, mutants of the Drosophila melanogaster ERC/CAST family protein Bruchpilot (BRP) were shown to lack dense projections (T-bars) and to suffer from Ca2+ channel–clustering defects. In this study, we used high resolution light microscopy, electron microscopy, and intravital imaging to analyze the function of BRP in AZ assembly. Consistent with truncated BRP variants forming shortened T-bars, we identify BRP as a direct T-bar component at the AZ center with its N terminus closer to the AZ membrane than its C terminus. In contrast, Drosophila Liprin-α, another AZ-organizing protein, precedes BRP during the assembly of newly forming AZs by several hours and surrounds the AZ center in few discrete punctae. BRP seems responsible for effectively clustering Ca2+ channels beneath the T-bar density late in a protracted AZ formation process, potentially through a direct molecular interaction with intracellular Ca2+ channel domains.

scholarly journals Homeostatic scaling of active zone scaffolds maintains global synaptic strength

2019 ◽  
Vol 218 (5) ◽  
pp. 1706-1724 ◽  
Author(s):  
Pragya Goel ◽  
Dominique Dufour Bergeron ◽  
Mathias A. Böhme ◽  
Luke Nunnelly ◽  
Martin Lehmann ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Axonal Transport ◽  
Active Zone ◽  
Synaptic Strength ◽  
Structure And Function ◽  
Synaptic Terminals ◽  
And Function

Synaptic terminals grow and retract throughout life, yet synaptic strength is maintained within stable physiological ranges. To study this process, we investigated Drosophila endophilin (endo) mutants. Although active zone (AZ) number is doubled in endo mutants, a compensatory reduction in their size homeostatically adjusts global neurotransmitter output to maintain synaptic strength. We find an inverse adaptation in rab3 mutants. Additional analyses using confocal, STED, and electron microscopy reveal a stoichiometric tuning of AZ scaffolds and nanoarchitecture. Axonal transport of synaptic cargo via the lysosomal kinesin adapter Arl8 regulates AZ abundance to modulate global synaptic output and sustain the homeostatic potentiation of neurotransmission. Finally, we find that this AZ scaling can interface with two independent homeostats, depression and potentiation, to remodel AZ structure and function, demonstrating a robust balancing of separate homeostatic adaptations. Thus, AZs are pliable substrates with elastic and modular nanostructures that can be dynamically sculpted to stabilize and tune both local and global synaptic strength.

scholarly journals Nanoscopical analysis reveals an orderly arrangement of the presynaptic scaffold protein Bassoon at the Golgi-apparatus

2021 ◽  
Author(s):  
Tina Ghelani ◽  
Carolina Montenegro ◽  
Anna Fejtova ◽  
Thomas Dresbach
Keyword(s):  
Golgi Apparatus ◽  
Active Zone ◽  
Hippocampal Neurons ◽  
Scaffold Protein ◽  
Stimulated Emission ◽  
Trans Golgi Network ◽  
C Terminus ◽  
N Terminus ◽  
Orderly Fashion ◽  
Golgi Network

Bassoon is a core scaffold protein of the presynaptic active zone. In brain synapses, the C-terminus of Bassoon is oriented toward the plasma membrane and its N-terminus is oriented towards synaptic vesicles. At the Golgi-apparatus Bassoon is thought to assemble active zone precursor structures, but whether it is arranged in an orderly fashion is unknown. Understanding the topology of this large scaffold protein is important for models of active zone biogenesis. Using stimulated emission depletion nanoscopy in cultured hippocampal neurons, we found that an N-terminal intramolecular tag of recombinant Bassoon, but not C-terminal tag, colocalized with markers of the trans-Golgi network. The N-terminus of Bassoon was located between 48 nm and 69 nm away from TGN38, while its C-terminus was located between 100 nm and 115 nm away from TGN38. Sequences within the first 95 amino acids of Bassoon were required for this arrangement. Our data are consistent with a model, in which the N-terminus of Bassoon binds to the membranes of the trans-Golgi network, while the C-terminus associates with active zone components, thus reflecting the topographic arrangement characteristic of synapses also at the Golgi-apparatus.

scholarly journals MxB restricts HIV-1 by targeting the tri-hexamer interface of the viral capsid

2018 ◽  
Author(s):  
Sarah Sierra Smaga ◽  
Chaoyi Xu ◽  
Brady James Summers ◽  
Katherine Marie Digianantonio ◽  
Juan Roberto Perilla ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Viral Genome ◽  
Specific Interaction ◽  
Md Simulations ◽  
Building Blocks ◽  
Binding Pocket ◽  
Restriction Factor ◽  
Antiviral Protein ◽  
N Terminus ◽  
Hiv 1

AbstractMyxovirus resistance protein B (MxB) is an interferon-inducible restriction factor of HIV-1 that blocks nuclear import of the viral genome. Evidence suggests that MxB recognizes higher-order interfaces of the HIV capsid lattice, but the mechanistic details of this interaction are not known. Previous studies have mapped the restriction activity of MxB to its N-terminus encompassing a triple arginine motif11RRR13. Here we demonstrate a direct and specific interaction between the MxB N-terminus and helical assemblies of HIV-1 capsid protein (CA) using highly purified recombinant proteins. We performed thorough mutagenesis to establish the detailed molecular requirements for the CA interaction with MxB. The results map MxB binding to the interface of three CA hexamers, specifically interactions between positively charged MxB N-terminal residues and negatively charged CA residues. Our crystal structures show that the CA mutations affecting MxB interaction and restriction do not alter the conformation of capsid assembly. In addition, 30 microsecond long all-atom molecular dynamics (MD) simulations of the complex between the MxB N-terminus and the HIV CA tri-hexamer interface show persistent MxB binding and identify a MxB-binding pocket surrounded by three CA hexamers. These results establish the molecular details of the binding of a lattice-sensing host factor onto HIV capsid, and provide insight into how MxB recognizes HIV capsid for the restriction of HIV-1 infection.Author summaryThe human antiviral protein MxB is a restriction factor that fights HIV infection. Previous experiments have demonstrated that MxB targets the HIV capsid, a protein shell that protects the viral genome. To make the conical shaped capsid, HIV CA proteins are organized into a lattice composed of hexamer and pentamer building blocks, providing many interfaces for host proteins to recognize. Through extensive biochemical and biophysical studies and molecular dynamics simulations, we show that MxB is targeting the HIV capsid by recognizing the region created at the intersection of three CA hexamers. We are further able to map this interaction to a few CA residues, located in a negatively-charged well at the interface between the three CA hexamers. This work provides detailed residue-level mapping of the targeted capsid interface and how MxB interacts. This information could inspire the development of capsid-targeting therapies for HIV.

scholarly journals Crossover recombination and synapsis are linked by adjacent regions within the N terminus of the Zip1 synaptonemal complex protein

2018 ◽  
Author(s):  
Karen Voelkel-Meiman ◽  
Shun-Yun Cheng ◽  
Melanie Parziale ◽  
Savannah J. Morehouse ◽  
Arden Feil ◽  
...  
Keyword(s):  
Synaptonemal Complex ◽  
Chromosome Segregation ◽  
Central Element ◽  
Building Blocks ◽  
Homologous Chromosomes ◽  
Sumo Ligase ◽  
Close Proximity ◽  
N Terminus ◽  
Complex Protein ◽  
Synaptonemal Complex Protein

AbstractAccurate chromosome segregation during meiosis relies on the prior establishment of at least one crossover recombination event between homologous chromosomes, which is often associated with the meiosis-specific MutSγ complex. The recombination intermediates that give rise to MutSγ interhomolog crossovers are embedded within a hallmark meiotic prophase structure called the synaptonemal complex (SC), but the mechanisms that coordinate the processes of SC assembly (synapsis) and crossover recombination remain poorly understood. Among known central region building blocks of the budding yeast SC, the Zip1 protein is unique for its SC-independent role in promoting MutSγ crossovers. Here we report that adjacent regions within Zip1’s unstructured N terminus encompass its crossover and SC assembly functions. We previously showed that deletion of Zip1 residues 21-163 abolishes tripartite SC assembly and prevents the robust SUMOylation of the SC central element component, Ecm11, but allows excess MutSγ crossover recombination. We find the reciprocal phenotype when Zip1 residues 2-9 or 10-14 are deleted; in these mutants SC assembles and Ecm11 is hyperSUMOylated, but MutSγ crossovers are strongly diminished. Interestingly, Zip1 residues 2-9 or 2-14 are required for the normal localization of Zip3, a putative E3 SUMO ligase and pro-MutSγ crossover factor, to Zip1 polycomplex structures and to recombination initiation sites. By contrast, deletion of Zip1 residues 15-20 does not detectably prevent Zip3’s localization at Zip1 polycomplex and supports some MutSγ crossing over but prevents normal SC assembly and robust Ecm11 SUMOylation. These results highlight distinct N terminal regions that are differentially critical for Zip1’s roles in crossover recombination and SC assembly; we speculate that the adjacency of these regions enables Zip1 to serve as a liaison, facilitating crosstalk between the two processes by bringing crossover recombination and synapsis factors in close proximity to one another.

scholarly journals Protein Modifications: From Chemoselective Probes to Novel Biocatalysts

Catalysts ◽  
2021 ◽  
Vol 11 (12) ◽  
pp. 1466
Author(s):  
Tomás Bohn Pessatti ◽  
Hernán Terenzi ◽  
Jean Borges Bertoldo
Keyword(s):  
Biomedical Applications ◽  
Building Blocks ◽  
Protein Scaffolds ◽  
Chemical Methods ◽  
Native Proteins ◽  
Naturally Occurring ◽  
N Terminus ◽  
Canonical Amino Acid ◽  
History Of ◽  
Rich History

Chemical reactions can be performed to covalently modify specific residues in proteins. When applied to native enzymes, these chemical modifications can greatly expand the available set of building blocks for the development of biocatalysts. Nucleophilic canonical amino acid sidechains are the most readily accessible targets for such endeavors. A rich history of attempts to design enhanced or novel enzymes, from various protein scaffolds, has paved the way for a rapidly developing field with growing scientific, industrial, and biomedical applications. A major challenge is to devise reactions that are compatible with native proteins and can selectively modify specific residues. Cysteine, lysine, N-terminus, and carboxylate residues comprise the most widespread naturally occurring targets for enzyme modifications. In this review, chemical methods for selective modification of enzymes will be discussed, alongside with examples of reported applications. We aim to highlight the potential of such strategies to enhance enzyme function and create novel semisynthetic biocatalysts, as well as provide a perspective in a fast-evolving topic.

scholarly journals Effects of modifications near the 2-, 3- and 4-fold symmetry axes on human ferritin renaturation

1997 ◽  
Vol 322 (2) ◽  
pp. 461-468 ◽  
Author(s):  
Paolo SANTAMBROGIO ◽  
Patrizia PINTO ◽  
Sonia LEVI ◽  
Anna COZZI ◽  
Ermanna ROVIDA ◽  
...  
Keyword(s):  
Guanidine Hydrochloride ◽  
Building Blocks ◽  
Fold Axis ◽  
Wild Type ◽  
Chemical Modifications ◽  
Specific Chemical ◽  
Large Hysteresis ◽  
N Terminus ◽  
Human Ferritin ◽  
Ferritin H

Ferritin is a protein of 24 subunits which assemble into a shell with 432 point symmetry. It can be denatured reversibly in acidic guanidine hydrochloride, with the formation of poorly populated renaturation intermediates. In order to increase the accumulation of intermediates and to study the mechanism of ferritin renaturation, we analysed variants of the human ferritin H-chain altered at the N-terminus (Δ1–13), near the 4-fold axis (Leu-169→Arg), the 3-fold axis (Asp-131→Ile+Glu-134→Phe) or the 2-fold axis (Ile-85→Cys). We also carried out specific chemical modifications of Cys-130 (near the 3-fold axis) and Cys-85 (near the 2-fold axis). Renaturation of the modified ferritins yielded assembly intermediates that differed in size and physical properties. Alterations of residues around the 2-, 4- and 3-fold axes produced subunit monomers, dimers and higher oligomers respectively. All these intermediates could be induced to assemble into ferritin 24-mers by concentrating them or by co-renaturing them with wild-type H-ferritin. The results support the hypothesis that the symmetric subunit dimers are the building blocks of ferritin assembly, and are consistent with a reassembly pathway involving the coalescence of dimers, probably around the 4-fold axis, followed by stepwise addition of dimers until the 24-mer cage is completed. In addition they show that assembly interactions are responsible for the large hysteresis of folding and unfolding plots. The implications of the studies for in vivoheteropolymer formation in vertebrates, which have two types of ferritin chain (H and L), are discussed.

Photochemical Evolution of Interstellar/Precometary Organic Material

1997 ◽  
Vol 161 ◽  
pp. 23-47 ◽  
Author(s):  
Louis J. Allamandola ◽  
Max P. Bernstein ◽  
Scott A. Sandford
Keyword(s):  
Origin Of Life ◽  
Building Blocks ◽  
Complex Species ◽  
Infrared Observations ◽  
Interstellar Ice ◽  
Polycyclic Aromatic ◽  
Ultraviolet Photolysis ◽  
The Origin Of Life ◽  
Simple Molecules ◽  
Complex Organic

AbstractInfrared observations, combined with realistic laboratory simulations, have revolutionized our understanding of interstellar ice and dust, the building blocks of comets. Since comets are thought to be a major source of the volatiles on the primative earth, their organic inventory is of central importance to questions concerning the origin of life. Ices in molecular clouds contain the very simple molecules H2O, CH3OH, CO, CO2, CH4, H2, and probably some NH3and H2CO, as well as more complex species including nitriles, ketones, and esters. The evidence for these, as well as carbonrich materials such as polycyclic aromatic hydrocarbons (PAHs), microdiamonds, and amorphous carbon is briefly reviewed. This is followed by a detailed summary of interstellar/precometary ice photochemical evolution based on laboratory studies of realistic polar ice analogs. Ultraviolet photolysis of these ices produces H2, H2CO, CO2, CO, CH4, HCO, and the moderately complex organic molecules: CH3CH2OH (ethanol), HC(= O)NH2(formamide), CH3C(= O)NH2(acetamide), R-CN (nitriles), and hexamethylenetetramine (HMT, C6H12N4), as well as more complex species including polyoxymethylene and related species (POMs), amides, and ketones. The ready formation of these organic species from simple starting mixtures, the ice chemistry that ensues when these ices are mildly warmed, plus the observation that the more complex refractory photoproducts show lipid-like behavior and readily self organize into droplets upon exposure to liquid water suggest that comets may have played an important role in the origin of life.

Laboratory and in-situ analysis of comet dust

1988 ◽  
Vol 46 ◽  
pp. 8-9
Author(s):  
D.E. Brownlee ◽  
A.L. Albee
Keyword(s):  
Electron Microscopy ◽  
Solar System ◽  
Laboratory Study ◽  
Isotopic Analysis ◽  
Building Blocks ◽  
Sem Analysis ◽  
Early Solar System ◽  
Cometary Material ◽  
Comet Dust

Comets are primitive, kilometer-sized bodies that formed in the outer regions of the solar system. Composed of ice and dust, comets are generally believed to be relic building blocks of the outer solar system that have been preserved at cryogenic temperatures since the formation of the Sun and planets. The analysis of cometary material is particularly important because the properties of cometary material provide direct information on the processes and environments that formed and influenced solid matter both in the early solar system and in the interstellar environments that preceded it.The first direct analyses of proven comet dust were made during the Soviet and European spacecraft encounters with Comet Halley in 1986. These missions carried time-of-flight mass spectrometers that measured mass spectra of individual micron and smaller particles. The Halley measurements were semi-quantitative but they showed that comet dust is a complex fine-grained mixture of silicates and organic material. A full understanding of comet dust will require detailed morphological, mineralogical, elemental and isotopic analysis at the finest possible scale. Electron microscopy and related microbeam techniques will play key roles in the analysis. The present and future of electron microscopy of comet samples involves laboratory study of micrometeorites collected in the stratosphere, in-situ SEM analysis of particles collected at a comet and laboratory study of samples collected from a comet and returned to the Earth for detailed study.

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