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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.

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Measurement of Endogenous MALT1 Activity
内源性MALT1活性的测定

癌症生物学 > 通用技术 > 生物化学试验 > 蛋白质分析
作者: Daniel Nagel
Daniel NagelAffiliation: Cellular Signal Integration, Helmholtz Zentrum München, Munich, Germany
Bio-protocol author page: a691
 and Daniel Krappmann
Daniel KrappmannAffiliation: Cellular Signal Integration, Helmholtz Zentrum München, Munich, Germany
For correspondence: daniel.krappmann@helmholtz-muenchen.de
Bio-protocol author page: a460
Vol 3, Iss 14, 7/20/2013, 4525 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.821

[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)

[Abstract] MALT1(粘膜相关淋巴组织蛋白1)是NF-kB驱动的淋巴细胞活化和不同B细胞淋巴瘤实体的发展和存活的重要衔接蛋白。此外,MALT1是半胱氨酸蛋白酶,其在结构上类似于半胱天冬酶,同时具有不同的底物偏好和激活机制。已显示MALT1的这种半胱氨酸蛋白酶活性对于侵袭性ABC-DLBCL(活化的B细胞型弥漫性大B细胞淋巴瘤)的最佳NF-kB活化和存活是至关重要的,其突出显示蛋白酶作为有吸引力的治疗靶标治疗不同的B细胞淋巴瘤和免疫性疾病如类风湿性关节炎或多发性硬化。在该协议中,我们描述了荧光切割测定,其可以用于测量内源性以及异位MALT1活性。为此,细胞MALT1需要通过抗体免疫沉淀从裂解的细胞中沉淀,随后与荧光底物肽孵育。已经开发了MALT1切割测定以直接测定MALT1在适应性免疫应答过程中以及淋巴恶性肿瘤中的病理信号传导中的活性谱。此外,MALT1活性测定已经成功地用于用小分子抑制剂监测细胞MALT1抑制。

Materials and Reagents

  1. Primary human and murine cells
  2. T-cell-lines (Jurkat)
  3. B-cell-lines: ABC-(TMD8, HBL1, OCI-Ly3, U2932, OCI-Ly10, RIVA) and GCB-DLBCL (Su-DHL-6, Su-DHL-4, BJAB)
  4. RPMI 1640 (Life Technologies, catalog number: 21875 )
  5. IMDM (Life Technologies, catalog number: 21056 )
  6. Fetal Bovine Serum (FBS) (Life Technologies, catalog number: 10270 )
  7. PMA (Phorbol 12-Myristat 13-Acetat) (Calbiochem, catalog number: 16561-29-8 )
  8. Lonomycin (Calbiochem, catalog number: 407950 )
  9. Anti-CD3/CD28 (Hit3a/CD28.2) (BD Heidelberg)
  10. IgG1 (R19-15) (BD Pharmingen, catalog number: 553440 )
  11. IgG2a (A85-1) (BD Pharmingen, catalog number: 553387 )
  12. MALT1 antibody (Santa Cruz, catalog number: H300 )
  13. Protein-G Sepharose (GE Healthcare, catalog number: 17-0618-01 )
  14. PBS (Life Technologies, catalog number: 14190 )
  15. Ac-LRSR-AMC (Peptides International, catalog number: MCA-3952-PI )
  16. Z-VRPR-FMK (ENZO Life Sciences, catalog number: ALX-260-166 )
  17. Complete protease inhibitor cocktail tablets (Roche, catalog number: 11836145001 )
  18. Mepazine (Hit2lead, catalog number: 5216177 )
  19. 384-well non-binding plates, black (Greiner Bio-one, catalog number: 781900 )
  20. Saccharose
  21. CHAPS
  22. HEPES
  23. Triton X-100
  24. Cellular lysis buffer (see Recipes)
  25. MALT1 cleavage buffer (see Recipes)

Equipment

  1. 37 °C 5% CO2 cell culture incubator
  2. 26 G syringe (Roth, catalog number: C718.1 )
  3. Centrifuge (Eppendorf, model: 5417R )
  4. Synergy II multiwell plate reader (Biotek)
  5. Rotary Mixer

Procedure

  1. To activate MALT1 plate 2.5 x 106 cells (Jurkat T-cells, primary T cells or PBMCs) and either stimulate with PMA/Ionomycin (200 ng/ml and 300 ng/ml final concentrations) in 10 ml of complete RPMI media in 25 cm2 cell culture flasks or with anti-CD3 and anti-CD28 antibodies (1 μg/ml final concentration each) in the presence of anti-IgG1/IgG2a coupling antibodies (0.5 μg/ml each) in 1 ml of complete RPMI media in 12-well for 30 min or treat with solvent (H2O) in the control. Cells with constitutive MALT1 activity (e.g. ABC-DLBCL) can be left untreated.
  2. As a negative control treat the above cells with MALT1 inhibitors, e.g. the tetrapeptide Z-VRPR-FMK (50 μM) or small molecule inhibitors like mepazine (10 μM) 3 - 6 h prior to stimulation and lysis.
  3. All 2.5 x 106 cells per reaction are then pelleted at 300 x g for 5 min.
  4. Removal of supernatant and lysis of the cells with cellular lysis buffer (500 μl) by rotating for 20 min on 4 °C in a 1.5 ml reagent tube.
  5. Centrifugation of the lysates for 10 min at 21,000 x g to remove cell debris.
  6. Incubation of all of the lysate with 700 ng MALT1 antibody over night at 4 °C on a rotary mixer.
  7. Incubation with 15 μl Protein-G sepharose beads (beads were washed and diluted 1:2 in PBS and equilibrated before usage) for 60 min at 4 °C on a rotary mixer.
  8. Beads are pelleted and washed 3 times with PBS at 300 x g for 2 min at 4 °C.
  9. In the last washing step all PBS is discarded via a syringe and 45 μl cleavage buffer is added to the beads.
  10. The beads are pelleted at 300 x g for 1 min afterwards re-suspended carefully and 49 μl of the suspension (beads and cleavage buffer) are subsequently transferred to one well of a 384-well assay plate.
  11. 1 μl of the fluorogenic substrate Ac-LRSR-AMC is then added to the wells in a final concentration of 20 μM and the plates are placed into the plate reader where they are shaked for 10 sec to mix the reagents.
  12. After an incubation of 30 min at 30 °C the release of AMC fluorescence due to MALT1 cleavage is measured at 360 nm excitation and 460 nm emission over a time course of 90 min.
  13. Specificity of MALT1 cleavage activity in vitro can be assessed by adding 5 nM of Z-VRPR-FMK to a separate control reaction (negative control).


    Figure 1. Fluorogenic MALT1 cleavage assay. Jurkat T-cells (2.5 x 106) were pre-incubated with Mepazine or DMSO for 4 h and subsequently stimulated with PMA/Ionomycin (P/I) or antiCD3/CD28 for 30 min or left untreated. After lysis of the cells MALT1 was precipitated and the catalytic activity was measured after addition of Ac-LRSR-AMC in a Synergy II plate reader over 60 min at 2 min intervals.

Recipes

  1. Cellular lysis buffer
    50 mM HEPES (pH 7.5)
    10% (v/v) Glycerin
    0.1% (v/v)Triton X-100
    1 mM Dithiothreitol (DTT)
    150 mM NaCl
    2 mM MgCl2
    1 complete protease inhibitor tablet per 50 ml
  2. MALT1 cleavage buffer
    50 mM MES (pH 7.0)
    150 mM NaCl
    10% (w/v) Saccharose
    0.1% (w/v) CHAPS
    1 M Sodium citrate
    10 mM DTT

Acknowledgments

This protocol was adapted from two previous publications (Kloo et al., 2011; Nagel et al., 2012). The work was supported by a grant of the ‘Deutsche Krebshilfe e.V.’ to DK.

References

  1. Kloo, B., Nagel, D., Pfeifer, M., Grau, M., Duwel, M., Vincendeau, M., Dorken, B., Lenz, P., Lenz, G. and Krappmann, D. (2011). Critical role of PI3K signaling for NF-kappaB-dependent survival in a subset of activated B-cell-like diffuse large B-cell lymphoma cells. Proc Natl Acad Sci U S A 108(1): 272-277.
  2. Nagel, D., Spranger, S., Vincendeau, M., Grau, M., Raffegerst, S., Kloo, B., Hlahla, D., Neuenschwander, M., Peter von Kries, J., Hadian, K., Dorken, B., Lenz, P., Lenz, G., Schendel, D. J. and Krappmann, D. (2012). Pharmacologic inhibition of MALT1 protease by phenothiazines as a therapeutic approach for the treatment of aggressive ABC-DLBCL. Cancer Cell 22(6): 825-837.

材料和试剂

  1. 原代人和鼠细胞
  2. T细胞系(Jurkat)
  3. B细胞系:ABC-(TMD8,HBL1,OCI-Ly3,U2932,OCI-Ly10,RIVA)和GCB-DLBCL(Su-DHL-6,Su-DHL-4,BJAB)
  4. RPMI 1640(Life Technologies,目录号:21875)
  5. IMDM(Life Technologies,目录号:21056)
  6. 胎牛血清(FBS)(Life Technologies,目录号:10270)
  7. PMA(Phorbol 12- Myristat 13-Acetat)(Calbiochem,目录号:16561-29-8)
  8. 洛美霉素(Calbiochem,目录号:407950)
  9. 抗CD3/CD28(Hit3a/CD28.2)(BD Heidelberg)
  10. IgG1(R19-15)(BD Pharmingen,目录号:553440)
  11. IgG2a(A85-1)(BD Pharmingen,目录号:553387)
  12. MALT1抗体(Santa Cruz,目录号:H300)
  13. 蛋白-G Sepharose(GE Healthcare,目录号:17-0618-01)
  14. PBS(Life Technologies,目录号:14190)
  15. Ac-LRSR-AMC(Peptides International,目录号:MCA-3952-PI)
  16. Z-VRPR-FMK(ENZO Life Sciences,目录号:ALX-260-166)
  17. 完全蛋白酶抑制剂混合物片剂(Roche,目录号:11836145001)
  18. 美吡嗪(Hit2lead,目录号:5216177)
  19. 384孔非结合板,黑色(Greiner Bio-one,目录号:781900)
  20. 蔗糖
  21. CHAPS
  22. HEPES
  23. Triton X-100
  24. 细胞裂解缓冲液(见配方)
  25. MALT1切割缓冲液(参见配方)

设备

  1. 37℃5%CO 2细胞培养孵育器
  2. 26 G注射器(Roth,目录号:C718.1)
  3. 离心机(Eppendorf,型号:5417R)
  4. Synergy II多孔板读数器(Biotek)
  5. 旋转搅拌机

程序

  1. 为了激活MALT1平板2.5×10 6个细胞(Jurkat T细胞,原代T细胞或PBMC),并用PMA /离子霉素刺激(200ng/ml 和300ng/ml的终浓度)在10毫升完全RPMI培养基中, 2抗体(每种0.5μg/ml)存在下,在1ml /孔细胞培养瓶中或用抗CD3和抗CD28抗体(各1μg/ml终浓度)的完全RPMI培养基在12孔中孵育30分钟或用对照中的溶剂(H 2 O)处理。具有组成型MALT1活性的细胞(例如ABC-DLBCL)可以不处理。
  2. 作为阴性对照,在刺激和裂解前3-6小时用MALT1抑制剂(例如四肽Z-VRPR-FMK(50μM)或小分子抑制剂如甲哌啶嗪(10μM))处理上述细胞。
  3. 然后将所有每个反应的2.5×10 6个细胞在300×g下沉淀5分钟。
  4. 通过在1.5ml试剂管中在4℃下旋转20分钟,除去上清液并用细胞裂解缓冲液(500μl)裂解细胞。
  5. 在21,000×g离心裂解物10分钟以除去细胞碎片。
  6. 所有裂解物用700ng MALT1抗体在4℃下在旋转混合器上孵育过夜。
  7. 用15μl蛋白-G琼脂糖珠(用PBS洗涤并稀释1:2并在使用前平衡)在旋转混合器上在4℃温育60分钟。
  8. 将珠粒沉淀并在4℃下以300xg的PBS用PBS洗涤3次,每次2分钟。
  9. 在最后的洗涤步骤中,通过注射器丢弃所有PBS,并向珠子中加入45μl切割缓冲液
  10. 将珠子以300×g离心1分钟,然后小心地重悬,随后将49μl悬浮液(珠子和裂解缓冲液)转移到384孔测定板的一个孔中。
  11. 然后向孔中加入1μl荧光底物Ac-LRSR-AMC,终浓度为20μM,将平板置于平板读数器中,在平板读数器中振荡10秒钟以混合试剂。
  12. 在30℃孵育30分钟后,在90分钟的时间过程中,在360nm激发和460nm发射下测量由于MALT1切割引起的AMC荧光的释放。
  13. 体外MALT1切割活性的特异性可通过将5nM Z-VRPR-FMK加入单独的对照反应(阴性对照)来评估。


    图1.荧光MALT1切割测定。 Jurkat T细胞(2.5×10 6)与甲哌啶嗪或DMSO预孵育4小时,随后用PMA /离子霉素(P/I)或抗CD3/CD28刺激30小时min或未处理。细胞裂解后,沉淀MALT1,在Synergy II读板器中以2分钟的间隔在60分钟内加入Ac-LRSR-AMC后测量催化活性。

食谱

  1. 细胞裂解缓冲液
    50mM HEPES(pH7.5) 10%(v/v)甘油 0.1%(v/v)Triton X-100 1mM二硫苏糖醇(DTT) 150mM NaCl 2mM MgCl 2/
    每50ml中加入1μg完全蛋白酶抑制剂片剂
  2. MALT1裂解缓冲液
    50mM MES(pH7.0) 150mM NaCl 10%(w/v)蔗糖 0.1%(w/v)CHAPS
    1 M柠檬酸钠
    10 mM DTT

致谢

该方案改编自两个以前的出版物(Kloo等人,2011; Nagel等人,2012)。 这项工作得到了德意志银行授予的"Deutsche Krebshilfe e.V."的支持。

参考文献

  1. Kloo,B.,Nagel,D.,Pfeifer,M.,Grau,M.,Duwel,M.,Vincendeau,M.,Dorken,B.,Lenz,P.,Lenz,G.and Krappmann, 2011)。 PI3K信号传导在NF-kappaB依赖性存活的一个子集中的关键作用激活的B细胞 - 例如弥漫性大B细胞淋巴瘤细胞。美国国家科学院院报108(1):272-277。
  2. Nagel,D.,Spranger,S.,Vincendeau,M.,Grau,M.,Raffegerst,S.,Kloo,B.,Hlahla,D.,Neuenschwander,M.,Peter von Kries,J.,Hadian,K 。,Dorken,B.,Lenz,P.,Lenz,G.,Schendel,DJ和Krappmann,D。(2012)。
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How to cite this protocol: Nagel, D. and Krappmann, D. (2013). Measurement of Endogenous MALT1 Activity. Bio-protocol 3(14): e821. DOI: 10.21769/BioProtoc.821; Full Text



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