内源性MALT1活性的测定

Daniel Nagel; Daniel Krappmann

doi:10.21769/BioProtoc.821

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Peer-reviewed

Measurement of Endogenous MALT1 Activity

内源性MALT1活性的测定

DN Daniel Nagel

Daniel Krappmann email

发布: 2013年07月20日第3卷第14期 DOI: 10.21769/BioProtoc.821 浏览次数: 12149

评审: Lin FangFanglian He

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参见作者原研究论文

The authors used this protocol in:

Cover of Cancer Cell, featuring study using the protocol.

Dec 2012

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Abstract

MALT1(Mucosa associated lymphoid tissue protein 1) is an important adapter protein for the NF-kB driven lymphocyte activation and the development and survival of distinct B-cell lymphoma entities. In addition MALT1 is a cysteine protease that structurally resembles caspases while having a different substrate preference and mechanism of activation. This paracaspase activity of MALT1 has been shown to be critical for an optimal NF-kB activation and survival of the aggressive ABC-DLBCL (Activated B cell-type of diffuse large B cell lymphoma), which highlights the protease as an attractive therapeutic target for the treatment of distinct B-cell lymphomas and immune diseases like rheumatoid arthritis or multiple sclerosis. In this protocol we describe a fluorogenic cleavage assay, which can be used to measure endogenous and also ectopic MALT1 activity. To this end, cellular MALT1 needs to be precipitated from the lysed cells via antibody immunoprecipitation and subsequently incubated with a fluorogenic substrate peptide. The MALT1 cleavage assay has been developed to directly determine the activity profile of MALT1 in the course of the adaptive immune response as well as in pathological signaling in lymphoid malignancies. In addition, the MALT1 activity assay has been successfully used to monitor cellular MALT1 inhibition with small molecule inhibitors.

Keywords: Protease (蛋白酶)

Paracaspase (paracaspase)

Immune signaling (免疫信号转导)

T cell activation (T细胞活化)

Lymphoma (淋巴瘤)

Materials and Reagents

Primary human and murine cells
T-cell-lines (Jurkat)
B-cell-lines: ABC-(TMD8, HBL1, OCI-Ly3, U2932, OCI-Ly10, RIVA) and GCB-DLBCL (Su-DHL-6, Su-DHL-4, BJAB)
RPMI 1640 (Life Technologies, catalog number: 21875 )
IMDM (Life Technologies, catalog number: 21056 )
Fetal Bovine Serum (FBS) (Life Technologies, catalog number: 10270 )
PMA (Phorbol 12-Myristat 13-Acetat) (Calbiochem, catalog number: 16561-29-8 )
Lonomycin (Calbiochem, catalog number: 407950 )
Anti-CD3/CD28 (Hit3a/CD28.2) (BD Heidelberg)
IgG1 (R19-15) (BD Pharmingen, catalog number: 553440 )
IgG2a (A85-1) (BD Pharmingen, catalog number: 553387 )
MALT1 antibody (Santa Cruz, catalog number: H300 )
Protein-G Sepharose (GE Healthcare, catalog number: 17-0618-01 )
PBS (Life Technologies, catalog number: 14190 )
Ac-LRSR-AMC (Peptides International, catalog number: MCA-3952-PI )
Z-VRPR-FMK (ENZO Life Sciences, catalog number: ALX-260-166 )
Complete protease inhibitor cocktail tablets (Roche, catalog number: 11836145001 )
Mepazine (Hit2lead, catalog number: 5216177 )
384-well non-binding plates, black (Greiner Bio-one, catalog number: 781900 )
Saccharose
CHAPS
HEPES
Triton X-100
Cellular lysis buffer (see Recipes)
MALT1 cleavage buffer (see Recipes)