In Vivo Protein Cross-Linking

Agou, Fabrice; Ye, Fei; Véron, Michel

doi:10.1385/1-59259-762-9:427

Cited by 12 publications

(12 citation statements)

References 18 publications

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“…On the other hand, molecular interactions between reggie-1 and reggie-2 have been examined using yeast two-hybrid assays, co-immunoprecipitation and colocalization [13,17,20]; however, no evidence for the formation of reggie hetero-oligomers has been provided so far. We assessed the oligomerization state of reggie proteins using chemical crosslinking, which is a powerful method to examine protein-protein interactions in living cells [27,28]. As reggies contain many lysine residues (34 and 30 in reggie-1 and -2 respectively), we used cell-permeable lysine-specific cross-linkers.…”

Section: Reggie Proteins Form Homo-and Hetero-tetramers In Living Cellsmentioning

confidence: 99%

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Solis

Hoegg

Munderloh

et al. 2007

Biochemical Journal

189

214

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Reggie-1 and -2 proteins (flotillin-2 and -1 respectively) form their own type of non-caveolar membrane microdomains, which are involved in important cellular processes such as T-cell activation, phagocytosis and signalling mediated by the cellular prion protein and insulin; this is consistent with the notion that reggie microdomains promote protein assemblies and signalling. While it is generally known that membrane microdomains contain large multiprotein assemblies, the exact organization of reggie microdomains remains elusive. Using chemical cross-linking approaches, we have demonstrated that reggie complexes are composed of homo- and hetero-tetramers of reggie-1 and -2. Moreover, native reggie oligomers are indeed quite stable, since non-cross-linked tetramers are resistant to 8 M urea treatment. We also show that oligomerization requires the C-terminal but not the N-terminal halves of reggie-1 and -2. Using deletion constructs, we analysed the functional relevance of the three predicted coiled-coil stretches present in the C-terminus of reggie-1. We confirmed experimentally that reggie-1 tetramerization is dependent on the presence of coiled-coil 2 and, partially, of coiled-coil 1. Furthermore, since depletion of reggie-1 by siRNA (small interfering RNA) silencing induces proteasomal degradation of reggie-2, we conclude that the protein stability of reggie-2 depends on the presence of reggie-1. Our data indicate that the basic structural units of reggie microdomains are reggie homo- and hetero-tetramers, which are dependent on the presence of reggie-1.

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Section: Reggie Proteins Form Homo-and Hetero-tetramers In Living Cellsmentioning

confidence: 99%

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Solis

Hoegg

Munderloh

et al. 2007

Biochemical Journal

189

214

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“…A thorough understanding of protein interactions and structures of protein complexes is fundamental to the understanding of protein function and regulation. Chemical cross-linking coupled with mass spectrometry (MS) is a powerful method that can be used to study protein-protein interactions [1–6]. The unique capability of chemical cross-linking to stabilize protein interactions through covalent bond formation allows not only the structural elucidation [7–17], but also the detection of stable, weak and/or transient protein-protein interactions in native cells or tissues [18–25].…”

Section: Introductionmentioning

confidence: 99%

Selective enrichment and identification of azide-tagged cross-linked peptides using chemical ligation and mass spectrometry

Vellucci

Kao

Kaake

et al. 2010

J. Am. Soc. Mass Spectrom.

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Protein-protein interaction is one of the key regulatory mechanisms for controlling protein function in various cellular processes. Chemical cross-linking coupled with mass spectrometry has proven to be a powerful method not only for mapping protein-protein interactions of all natures, including weak and transient ones, but also for determining their interaction interfaces. One critical challenge remaining in this approach is how to effectively isolate and identify cross-linked products from a complex peptide mixture. In this work, we have developed a novel strategy using conjugation chemistry for selective enrichment of cross-linked products. An azide-tagged cross-linker along with two biotinylated conjugation reagents were designed and synthesized. Cross-linking of model peptides and cytochrome c as well as enrichment of the resulting cross-linked peptides has been assessed. Selective conjugation of azide-tagged cross-linked peptides has been demonstrated using two strategies: copper catalyzed cycloaddition and Staudinger ligation. While both methods are effective, Staudinger ligation is better suited for enriching the cross-linked peptides since there are fewer issues with sample handling. LC MS n analysis coupled with database searching using the Protein Prospector software package allowed identification of 58 cytochrome c cross-linked peptides after enrichment and affinity purification. The new enrichment strategy developed in this work provides useful tools for facilitating identification of cross-linked peptides in a peptide mixture by MS, thus presenting a step forward in future studies of protein-protein interactions of protein complexes by cross-linking and mass spectrometry. (J Am Soc Mass Spectrom 2010, 21, 1432-1445) © 2010 American Society for Mass Spectrometry P roteins form stable and/or dynamic multisubunit protein complexes under different physiologic conditions to maintain cell viability and normal cell homeostasis. A thorough understanding of protein interactions and structures of protein complexes is fundamental to the understanding of protein function and regulation. Chemical cross-linking coupled with mass spectrometry (MS) is a powerful method that can be used to study protein-protein interactions [1][2][3][4][5][6]. The unique capability of chemical cross-linking to stabilize protein interactions through covalent bond formation allows not only the structural elucidation [7][8][9][10][11][12][13][14][15][16][17], but also the detection of stable, weak, and/or transient protein-protein interactions in native cells or tissues [18 -25].In addition to capturing protein interacting partners, many studies have shown that chemical cross-linking can yield low-resolution structural information about the constraints within a molecule [2][3][4]. Traditional methods such as NMR analysis and X-ray crystallography can yield detailed information on protein structure, however NMR spectroscopy requires large quantities of pure protein in a specific solvent, and X-ray crystallography is often limited by th...

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“…NEMO is able to be self‐multimerized, either in a trimerization form or a tetramerization form through the LZ and C‐terminal coiled‐coil domain (32–34). The ZF itself is not involved in the multimerization since deleting the ZF does not significantly affect the multimerization of NEMO (35). However, the extra ZF that added on to NEMO dimer via multimerization might assist the LZ to recruit upstream factor(s) to promote NF‐κB activation (32).…”

Section: Early‐phase Activation Of Nf‐κb After Uvcmentioning

confidence: 99%

Differential Signaling Circuits in Regulation of Ultraviolet C Light‐induced Early‐ and Late‐phase Activation of NF‐κB

Tong

2010

Photochem & Photobiology

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Ultraviolet C light (UVC) induces nuclear factor‐kappa B (NF‐κB) activation via a complex network. In the early phase (4–12 h) of irradiation, NF‐κB activation is accompanied with IκBα reduction via a translation inhibition pathway. In the late phase of UVC‐induced NF‐κB activation (16–24 h), the IκBα depletion is a combined result of regulation at both transcriptional and translational levels. However, the NF‐κB activation appears to be independent of the level of IκBα. In this review, we will discuss the multiple signaling circuits that regulate NF‐κB activation during the early and late phases of UVC irradiation.

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In Vivo Protein Cross-Linking

Cited by 12 publications

References 18 publications

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Reggie/flotillin proteins are organized into stable tetramers in membrane microdomains

Selective enrichment and identification of azide-tagged cross-linked peptides using chemical ligation and mass spectrometry

Differential Signaling Circuits in Regulation of Ultraviolet C Light‐induced Early‐ and Late‐phase Activation of NF‐κB

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