Method for identifying ubiquitin and ubiquitin-like enzyme activities
AssigneesCentre National De La Recherche Scientifique, Universite De Montpellier
InventorsGuillaume Bossis, Pierre Gatel, Marc Piechaczyk
Life of this patent
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Abstract
A method for quantifying the activity of the proteins/enzymes involved in the conjugation of the SUMO/Ubiquitin/Nedd8 proteins in a cell of a biological sample the method including: a) a step of contacting a cellular extract of cell with each protein of a subgroup of at least 3 proteins wherein the at least 3 proteins corresponds to the proteins essentially including or only including the sequences SEQ ID NO: 1 to 3, b) a step of simultaneously measuring ubiquitination, sumoylation and neddylation level of each of the at least 3 proteins to obtain a first value for ubiquitin, SUMO and Nedd8.
Claims9
A method for quantifying the activity of the proteins or enzymes involved in the conjugation of the SUMO/Ubiquitin/Nedd8 proteins in a cell of a biological sample, comprising:
- a) contacting a cellular extract of the cell of the biological sample with a composition comprising at least 3 proteins,
- SEQ ID NOs:
- 1 to 3,
- wherein each of said at least 3 proteins is supported by a distinct type of fluorescent bead
- that has a distinct fluorescent property,
- wherein said at least 3 proteins are not conjugated by SUMO/Ubiquitin/Nedd8 proteins before contacting said cellular extract, and
- wherein SEQ ID NO:
- 1 corresponds to ZMYM5 protein;
- SEQ ID NO:
- 2 corresponds to BEAN protein;
- and SEQ ID NO:
- 3 corresponds to OTUD6B protein;
- and b) simultaneously measuring ubiquitination of BEAN of SEQ ID NO:
- 2, SUMOylation of ZMYM5 of SEQ ID NO:
- 1, and NEDDylation of OTUD6B of SEQ ID NO:
- 3 to obtain first values of activity levels for ubiquitin, SUMO and Nedd8.
A method for identifying the effect of a drug on protein modification by SUMO/Ubiquitin/Nedd8 proteins in a cell of a biological sample, comprising:
- a) treating a portion of the biological sample with a drug,
- b) contacting a cellular extract of the drug-treated portion of the biological sample with a composition comprising at least 3 proteins
- corresponding to SEQ ID NOs:
- 1 to 3,
- c) contacting a cellular extract of a non-drug-treated portion of the biological sample with said composition,
- wherein each of said at least 3 proteins is supported by a distinct type of fluorescent bead
- that has a distinct fluorescent property,
- wherein said at least 3 proteins are not conjugated by SUMO/Ubiquitin/Nedd8 proteins before contacting said cellular extract, and
- wherein SEQ ID NO:
- 1 corresponds to ZMYM5 protein;
- SEQ ID NO:
- 2 corresponds to BEAN protein;
- and SEQ ID NO:
- 3 corresponds to OTUD6B protein;
- d) simultaneously measuring ubiquitination of BEAN of SEQ ID NO:2, SUMOylation of ZMYM5 of SEQ ID NO:
- 1, and NEDDylation of OTUD6B of SEQ ID NO:
- 3 in the cellular extract of the drug-treated portion of the biological sample to obtain first values of activity levels for ubiquitin, SUMO and Nedd8 and in the cellular extract of the non-drug-treated portion of biological sample to obtain second values of activity levels for ubiquitin, SUMO, and Nedd8;
- e) comparing the first values obtained to the second values to obtain a ratio between the first values and the second values;
- and
- f) determining that
- i. if the ratio is equal to 1, then said drug has no effect on protein modification by SUMO/Ubiquitin/Nedd8 proteins, and
- ii. if the ratio is not equal to 1, then said drug affects protein modification by SUMO/Ubiquitin/Nedd8 proteins.
A composition comprising at least 3 proteins;
- SEQ ID NOs:
- 1 to 3,
- wherein each of said at least 3 proteins is supported by a distinct fluorescent bead;
- that has a distinct fluorescent property.
Dependencies are inferred from the claim text.