1D continuous gel electrophoresis composition for the separation of deamidated proteins

Boudier-Lemosquet, Axel; Mahler, Adrien; Bobo, Claude; Moreau, Violaine; Priault, Muriel

doi:10.1016/j.ymeth.2021.03.002

Cited by 4 publications

(6 citation statements)

References 37 publications

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“…It is now well established that Bcl-xL is the target of nonenzymatic deamidation on residues N52 and N66 located in the unstructured loop between α1 and α2 helices [ 29 , 30 ]. Cell-free protein synthesis conditions are likely favorable for Bcl-xL deamidation [ 37 ], because of pH (8.0), temperature (28 °C), and duration (16 h).…”

Section: Resultsmentioning

confidence: 99%

“…Recombinant Bcl-xL expressed in E. coli was purified from inclusion bodies, according to [ 21 , 37 ]. The protein was stored in Tris/HCl 20 mM (pH 8.0), NaCl 450 mM, and DTT 10 mM.…”

Section: Methodsmentioning

confidence: 99%

“…( A ) Different fractions of recombinant Bcl-xL were loaded on isoelectrofocusing (IEF) gels (Thermofisher), according to the manufacturer’s instructions. From left to right: Bcl-xL extraction from inclusion bodies in E. coli [ 21 , 37 ]; Bcl-xL produced in cell-free system, inserted into nanodiscs (fraction “b” in Figure 1 B); Bcl-xL co-produced in cell-free system with its partner Bax, without nanodiscs [ 34 ]; Bcl-xL produced in cell-free system, not inserted into nanodiscs (fraction “u” in Figure 1 B); Bcl-xL in the soluble lysate from E. coli (classical method, before [ 21 ]). The blot is representative of two independent experiments.…”

Section: Figurementioning

confidence: 99%

“…The “E” fraction contained Bcl-xL but not His 7 -MSP1E3D1. ( B ) MALDI-MS analysis of the “E” fraction from above ( top ), compared to full-length Bcl-xL purified from inclusion bodies of E. coli [ 21 , 37 ] ( bottom ). Nanodisc-inserted Bcl-xL displayed a major peak at 26,046 Da (±10 Da) (mean of two individual measurements), close to the theoretical mass of full-length Bcl-xL (26,049 Da), while Bcl-xL purified from inclusion bodies displayed two peaks at 25,906 Da and 26,095 Da (mean of two individual measurements), close to the theoretical mass of full-length Bcl-xL deprived of the initiation methionine (theoretical mass of 25,917 Da) and to the full-length protein carrying an acetylated methionine (theoretical mass of 26,091 Da), respectively.…”

Section: Figurementioning

confidence: 99%

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Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System

et al. 2023

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The antiapoptotic protein Bcl-xL is a major regulator of cell death and survival, but many aspects of its functions remain elusive. It is mostly localized in the mitochondrial outer membrane (MOM) owing to its C-terminal hydrophobic α-helix. In order to gain further information about its membrane organization, we set up a model system combining cell-free protein synthesis and nanodisc insertion. We found that, contrary to its proapoptotic partner Bax, neosynthesized Bcl-xL was spontaneously inserted into nanodiscs. The deletion of the C-terminal α-helix of Bcl-xL prevented nanodisc insertion. We also found that nanodisc insertion protected Bcl-xL against the proteolysis of the 13 C-terminal residues that occurs during expression of Bcl-xL as a soluble protein in E. coli. Interestingly, we observed that Bcl-xL increased the insertion of Bax into nanodiscs, in a similar way to that which occurs in mitochondria. Cell-free synthesis in the presence of nanodiscs is, thus, a suitable model system to study the molecular aspects of the interaction between Bcl-xL and Bax during their membrane insertion.

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Section: Resultsmentioning

confidence: 99%

“…Recombinant Bcl-xL expressed in E. coli was purified from inclusion bodies, according to [ 21 , 37 ]. The protein was stored in Tris/HCl 20 mM (pH 8.0), NaCl 450 mM, and DTT 10 mM.…”

Section: Methodsmentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System

et al. 2023

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“…For the past 15 years, she has dissected the intrinsic instability that drives Bcl-xL deamidation. She pioneered the description of a monodeamidated form of Bcl-xL, and of the sequential mechanism that leads to the doubly deamidated form [ 167 , 168 ]. She has now specialized her lab on protein instability and molecular aging [ 169 ], and uses Bcl-xL deamidation both as a tool and as a study object.…”

Section: Team Workmentioning

confidence: 99%

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network

et al. 2022

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Since the Nobel Prize award more than twenty years ago for discovering the core apoptotic pathway in C. elegans, apoptosis and various other forms of regulated cell death have been thoroughly characterized by researchers around the world. Although many aspects of regulated cell death still remain to be elucidated in specific cell subtypes and disease conditions, many predicted that research into cell death was inexorably reaching a plateau. However, this was not the case since the last decade saw a multitude of cell death modalities being described, while harnessing their therapeutic potential reached clinical use in certain cases. In line with keeping research into cell death alive, francophone researchers from several institutions in France and Belgium established the French Cell Death Research Network (FCDRN). The research conducted by FCDRN is at the leading edge of emerging topics such as non-apoptotic functions of apoptotic effectors, paracrine effects of cell death, novel canonical and non-canonical mechanisms to induce apoptosis in cell death-resistant cancer cells or regulated forms of necrosis and the associated immunogenic response. Collectively, these various lines of research all emerged from the study of apoptosis and in the next few years will increase the mechanistic knowledge into regulated cell death and how to harness it for therapy.

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Introducing protein deamidation: Landmark discoveries, societal outreach, and tentative priming workflow to address deamidation

Boudier-Lemosquet

Mahler²,

Bobo³

et al. 2022

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1D continuous gel electrophoresis composition for the separation of deamidated proteins

Cited by 4 publications

References 37 publications

Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System

Bcl-xL Is Spontaneously Inserted into Preassembled Nanodiscs and Stimulates Bax Insertion in a Cell-Free Protein Synthesis System

Keeping Cell Death Alive: An Introduction into the French Cell Death Research Network

Introducing protein deamidation: Landmark discoveries, societal outreach, and tentative priming workflow to address deamidation

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