scholarly journals The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

2020 ◽  
Author(s):  
Alan Rodriguez Carvajal ◽  
Carlos Gomez Diaz ◽  
Antonia Vogel ◽  
Adar Sonn-Segev ◽  
Katrin Schodl ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Catalytic Activity ◽  
Ubiquitin Ligase ◽  
Bond Formation ◽  
Mass Spectrometry Data ◽  
Cross Linking ◽  
Concerted Action ◽  
Ubiquitin Chain ◽  
C Terminus

AbstractThe linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase that generates linear/Met1-linked ubiquitin chains. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between the C-terminus of ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. We present the first 3D reconstruction of LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that addition of the oxyester-bound branches depends on HOIL-1L catalytic activity. We suggest a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases supported by cross-linking mass spectrometry data, which show proximity between the catalytic RBR domains. Mutations in the linear ubiquitin chain-binding NZF domain of HOIL-1L reduces chain branching confirming its role in the process. In cells, these heterotypic chains were induced by TNF. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains with linear and oxyester-linked branches by the concerted action of HOIP and HOIL-1L.

scholarly journals The linear ubiquitin chain assembly complex LUBAC generates heterotypic ubiquitin chains

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Alan Rodriguez Carvajal ◽  
Irina Grishkovskaya ◽  
Carlos Gomez Diaz ◽  
Antonia Vogel ◽  
Adar Sonn-Segev ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Catalytic Activity ◽  
Ubiquitin Ligase ◽  
Bond Formation ◽  
Cross Linking ◽  
Concerted Action ◽  
Mouse Embryonic Fibroblasts ◽  
Embryonic Fibroblasts ◽  
Ubiquitin Chain

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RBR domains, a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.

scholarly journals CLMSVault: A Software Suite for Protein Cross-Linking Mass-Spectrometry Data Analysis and Visualization

2017 ◽  
Vol 16 (7) ◽  
pp. 2645-2652 ◽  
Author(s):  
Mathieu Courcelles ◽  
Jasmin Coulombe-Huntington ◽  
Émilie Cossette ◽  
Anne-Claude Gingras ◽  
Pierre Thibault ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Data Analysis ◽  
Mass Spectrometry Data ◽  
Cross Linking ◽  
Software Suite ◽  
Protein Cross Linking

Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes

2021 ◽  
pp. 217-232
Author(s):  
Lucía Quintana-Gallardo ◽  
Moisés Maestro-López ◽  
Jaime Martín-Benito ◽  
Miguel Marcilla ◽  
Daniel Rutz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Structural Characterization ◽  
Protein Complexes ◽  
Cross Linking

scholarly journals Mass spectrometry reveals the chemistry of formaldehyde cross-linking in structured proteins

2019 ◽  
Author(s):  
Tamar Tayri-Wilk ◽  
Moriya Slavin ◽  
Joanna Zamel ◽  
Ayelet Blass ◽  
Shon Cohen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Carbon Atom ◽  
Mass Spectrometry Data ◽  
Cross Linking ◽  
Atomic Structures ◽  
Cross Links ◽  
Structured Proteins ◽  
Structural Insights ◽  
Current Mechanism

AbstractFormaldehyde is a widely used fixative in biology and medicine. The current mechanism of formaldehyde cross-linking of proteins is the formation of a methylene bridge that incorporates one carbon atom into the link. Here, we present mass spectrometry data that largely refute this mechanism. Instead, the data reveal that cross-linking of structured proteins mainly involves a reaction that incorporates two carbon atoms into the link. Under MS/MS fragmentation, the link cleaves symmetrically to yield previously unrecognized fragments carrying a modification of one carbon atom. If these characteristics are considered, then formaldehyde cross-linking is readily applicable to the structural approach of cross-linking coupled to mass spectrometry. Using a cross-linked mixture of purified proteins, a suitable analysis identifies tens of cross-links that fit well with their atomic structures. A more elaborate in situ cross-linking of human cells in culture identified 469 intra-protein and 90 inter-protein cross-links, which also agreed with available atomic structures. Interestingly, many of these cross-links could not be mapped onto a known structure and thus provide new structural insights. For example, two cross-links involving the protein βNAC localize its binding site on the ribosome. Also of note are cross-links of actin with several auxiliary proteins for which the structure is unknown. Based on these findings we suggest a revised chemical reaction, which has relevance to the reactivity and toxicity of formaldehyde.

scholarly journals Structural prediction of protein models using distance restraints derived from cross-linking mass spectrometry data

2018 ◽  
Vol 13 (3) ◽  
pp. 478-494 ◽  
Author(s):  
Zsuzsanna Orbán-Németh ◽  
Rebecca Beveridge ◽  
David M Hollenstein ◽  
Evelyn Rampler ◽  
Thomas Stranzl ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Mass Spectrometry Data ◽  
Cross Linking ◽  
Structural Prediction ◽  
Distance Restraints ◽  
Protein Models

scholarly journals Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex

2018 ◽  
Vol 115 (42) ◽  
pp. E9792-E9801 ◽  
Author(s):  
Saikat Chowdhury ◽  
Chinatsu Otomo ◽  
Alexander Leitner ◽  
Kazuto Ohashi ◽  
Ruedi Aebersold ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Lipid Membranes ◽  
De Novo ◽  
Cross Linking ◽  
Double Membrane ◽  
Membrane Compartments ◽  
Biochemical Analyses ◽  
Chemical Cross Linking

Autophagy is an enigmatic cellular process in which double-membrane compartments, called “autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER–phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER–phagophore association and/or tethers vesicles to the ER–phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.

scholarly journals Mounting, structure and autocleavage of a type VI secretion-associated Rhs polymorphic toxin

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Dukas Jurėnas ◽  
Leonardo Talachia Rosa ◽  
Martial Rey ◽  
Julia Chamot-Rooke ◽  
Rémi Fronzes ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Spike Protein ◽  
Modular Organization ◽  
Cross Linking ◽  
Multidomain Proteins ◽  
Type Vi Secretion ◽  
Cryo Electron Microscopy ◽  
Dependent Growth ◽  
Contact Dependent Growth Inhibition

AbstractBacteria have evolved toxins to outcompete other bacteria or to hijack host cell pathways. One broad family of bacterial polymorphic toxins gathers multidomain proteins with a modular organization, comprising a C-terminal toxin domain fused to a N-terminal domain that adapts to the delivery apparatus. Polymorphic toxins include bacteriocins, contact-dependent growth inhibition systems, and specialized Hcp, VgrG, PAAR or Rhs Type VI secretion (T6SS) components. We recently described and characterized Tre23, a toxin domain fused to a T6SS-associated Rhs protein in Photorhabdus laumondii, Rhs1. Here, we show that Rhs1 forms a complex with the T6SS spike protein VgrG and the EagR chaperone. Using truncation derivatives and cross-linking mass spectrometry, we demonstrate that VgrG-EagR-Rhs1 complex formation requires the VgrG C-terminal β-helix and the Rhs1 N-terminal region. We then report the cryo-electron-microscopy structure of the Rhs1-EagR complex, demonstrating that the Rhs1 central region forms a β-barrel cage-like structure that encapsulates the C-terminal toxin domain, and provide evidence for processing of the Rhs1 protein through aspartyl autoproteolysis. We propose a model for Rhs1 loading on the T6SS, transport and delivery into the target cell.

scholarly journals Analysis of the Dynamic Proteasome Structure by Cross-Linking Mass Spectrometry

Biomolecules ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 505
Author(s):  
Marta L. Mendes ◽  
Gunnar Dittmar
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Structural Biology ◽  
26S Proteasome ◽  
Cross Linking ◽  
Macromolecular Complex ◽  
Dynamic Nature ◽  
Cryo Electron Microscopy ◽  
Integrative Structural Biology ◽  
Regulatory Particle

The 26S proteasome is a macromolecular complex that degrades proteins maintaining cell homeostasis; thus, determining its structure is a priority to understand its function. Although the 20S proteasome’s structure has been known for some years, the highly dynamic nature of the 19S regulatory particle has presented a challenge to structural biologists. Advances in cryo-electron microscopy (cryo-EM) made it possible to determine the structure of the 19S regulatory particle and showed at least seven different conformational states of the proteasome. However, there are still many questions to be answered. Cross-linking mass spectrometry (CLMS) is now routinely used in integrative structural biology studies, and it promises to take integrative structural biology to the next level, answering some of these questions.

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