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1.
Elife ; 102021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34142657

RESUMO

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, heme-oxidized IRP2 ubiquitin ligase 1 (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 RING-in-between-RING (RBR) domains, a coordinated ubiquitin relay mechanism between the HOIL-1-interacting protein (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.


Assuntos
Fatores de Transcrição , Ubiquitina-Proteína Ligases , Ubiquitina , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Feminino , Fibroblastos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Domínios Proteicos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
2.
EMBO J ; 39(3): e101625, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31556459

RESUMO

Meiosis is key to sexual reproduction and genetic diversity. Here, we show that the Arabidopsis cyclin-dependent kinase Cdk1/Cdk2 homolog CDKA;1 is an important regulator of meiosis needed for several aspects of meiosis such as chromosome synapsis. We identify the chromosome axis protein ASYNAPTIC 1 (ASY1), the Arabidopsis homolog of Hop1 (homolog pairing 1), essential for synaptonemal complex formation, as a target of CDKA;1. The phosphorylation of ASY1 is required for its recruitment to the chromosome axis via ASYNAPTIC 3 (ASY3), the Arabidopsis reductional division 1 (Red1) homolog, counteracting the disassembly activity of the AAA+ ATPase PACHYTENE CHECKPOINT 2 (PCH2). Furthermore, we have identified the closure motif in ASY1, typical for HORMA domain proteins, and provide evidence that the phosphorylation of ASY1 regulates the putative self-polymerization of ASY1 along the chromosome axis. Hence, the phosphorylation of ASY1 by CDKA;1 appears to be a two-pronged mechanism to initiate chromosome axis formation in meiosis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Sítios de Ligação , Cromossomos de Plantas/genética , Cromossomos de Plantas/metabolismo , Quinases Ciclina-Dependentes/genética , Proteínas de Ligação a DNA/química , Mutação , Fosforilação , Ligação Proteica , Multimerização Proteica
3.
Nat Protoc ; 13(7): 1724, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29942004

RESUMO

In the version of this article initially published online, the authors used incorrectly defined restraints for specifying the distance between residues when using the HADDOCK portal. Following the publication of a Correspondence by the developers of the HADDOCK portal (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0017-6, 2018) and a Reply by the authors of the Protocol (Nat. Protoc. https://dx.doi.org/10.1038/s41596-018-0018-5, 2018), the syntax in step 21 has been corrected. In addition, the input files (available as Supplementary Data 5-7) have been replaced.

5.
Nat Protoc ; 13(3): 478-494, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29419816

RESUMO

This protocol describes a workflow for creating structural models of proteins or protein complexes using distance restraints derived from cross-linking mass spectrometry experiments. The distance restraints are used (i) to adjust preliminary models that are calculated on the basis of a homologous template and primary sequence, and (ii) to select the model that is in best agreement with the experimental data. In the case of protein complexes, the cross-linking data are further used to dock the subunits to one another to generate models of the interacting proteins. Predicting models in such a manner has the potential to indicate multiple conformations and dynamic changes that occur in solution. This modeling protocol is compatible with many cross-linking workflows and uses open-source programs or programs that are free for academic users and do not require expertise in computational modeling. This protocol is an excellent additional application with which to use cross-linking results for building structural models of proteins. The established protocol is expected to take 6-12 d to complete, depending on the size of the proteins and the complexity of the cross-linking data.


Assuntos
Previsões/métodos , Espectrometria de Massas/métodos , Estrutura Terciária de Proteína/fisiologia , Simulação por Computador , Reagentes de Ligações Cruzadas/química , Modelos Moleculares , Estrutura Terciária de Proteína/genética , Proteínas/genética , Proteínas/fisiologia
7.
J Proteome Res ; 14(12): 5048-62, 2015 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-26535604

RESUMO

The HOP2-MND1 heterodimer is essential for meiotic homologous recombination in plants and other eukaryotes and promotes the repair of DNA double-strand breaks. We investigated the conformational flexibility of HOP2-MND1, important for understanding the mechanistic details of the heterodimer, with chemical cross-linking in combination with mass spectrometry (XL-MS). The final XL-MS workflow encompassed the use of complementary cross-linkers, quenching, digestion, size exclusion enrichment, and HCD-based LC-MS/MS detection prior to data evaluation. We applied two different homobifunctional amine-reactive cross-linkers (DSS and BS(2)G) and one zero-length heterobifunctional cross-linker (EDC). Cross-linked peptides of four biological replicates were analyzed prior to 3D structure prediction by protein threading and protein-protein docking for cross-link-guided molecular modeling. Miniaturization of the size-exclusion enrichment step reduced the required starting material, led to a high amount of cross-linked peptides, and allowed the analysis of replicates. The major interaction site of HOP2-MND1 was identified in the central coiled-coil domains, and an open colinear parallel arrangement of HOP2 and MND1 within the complex was predicted. Moreover, flexibility of the C-terminal capping helices of both complex partners was observed, suggesting the coexistence of a closed complex conformation in solution.


Assuntos
Proteínas de Arabidopsis/química , Fosfotransferases/química , Proteínas de Arabidopsis/metabolismo , Carbodi-Imidas/química , Cromatografia em Gel , Reagentes de Ligações Cruzadas/química , Modelos Moleculares , Complexos Multiproteicos/química , Fosfotransferases/metabolismo , Conformação Proteica , Multimerização Proteica , Succinimidas/química , Espectrometria de Massas em Tandem , Fluxo de Trabalho
8.
Biomol NMR Assign ; 8(1): 123-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23339032

RESUMO

Microtubule-associated protein 1B (MAP1B) is a classical high molecular mass microtubule-associated protein expressed at high levels in the brain. It confers specific properties to neuronal microtubules and is essential for neuronal differentiation, brain development and synapse maturation. Misexpression of the protein contributes to the development of brain disorders in humans. However, despite numerous reports demonstrating the importance of MAP1B in regulation of the neuronal cytoskeleton during neurite extension and axon guidance, its mechanism of action is still elusive. Here we focus on the intrinsically disordered microtubule binding domain of the light chain of MAP1B. In order to obtain more detailed structural information about this domain we assigned NMR chemical shifts of backbone and aliphatic side chain atoms.


Assuntos
Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Ressonância Magnética Nuclear Biomolecular , Sequência de Aminoácidos , Animais , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos
9.
J Biol Chem ; 280(3): 2257-65, 2005 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-15528209

RESUMO

The related high molecular mass microtubule-associated proteins (MAPs) MAP1A and MAP1B are predominantly expressed in the nervous system and are involved in axon guidance and synaptic function. MAP1B is implicated in fragile X mental retardation, giant axonal neuropathy, and ataxia type 1. We report the functional characterization of a novel member of the microtubule-associated protein 1 family, which we termed MAP1S (corresponding to sequence data bank entries for VCY2IP1 and C19ORF5). MAP1S contains the three hallmark domains of the microtubule-associated protein 1 family but hardly any additional sequences. It decorates neuronal microtubules and copurifies with tubulin from brain. MAP1S is synthesized as a precursor protein that is partially cleaved into heavy and light chains in a tissue-specific manner. Heavy and light chains interact to form the MAP1S complex. The light chain binds, bundles, and stabilizes microtubules and binds to actin. The heavy chain appears to regulate light chain activity. In contrast to MAP1A and MAP1B, MAP1S is expressed in a wide range of tissues in addition to neurons and represents the non-neuronal counterpart of this cytolinker family.


Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Actinas/metabolismo , Animais , Sequência de Bases , Encéfalo/crescimento & desenvolvimento , Primers do DNA , DNA Complementar , Genoma , Humanos , Imuno-Histoquímica , Camundongos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Peso Molecular , Células de Purkinje/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima
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