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Mucosa-associated-lymphoid-tissue (MALT) lymphoma of stomach is the most common extranodal lymphomas of human. Gastric MALT lymphoma is generally associated with H. pylori; and the eradication of this gastric infection results in durable tumor remission in 70% of the patients. Early identification of those patients in whom this approach is unlikely to be successful is a challenge to the clinicians. We, along with several distinguished groups, found that aberrant nuclear BCL10 expression is predictive of H. pylori-independent status in gastric MALT lymphoma. BCL10 normally resides in the cytoplasm and participates in signals to activate NF-B. The activation of NF-B is often associated with BCL10 phosphorylation. We started with the intentions listed below.
1. elucidating mechanism(s) involved in mediating the aberrant nuclear BCL10 expression.
Evidences indicated that aberrant nuclear localization of BCL10 might simply be due to interruption of its ability to self-associate or interact with cytoplasmic partners such as MALT1. In the process of studying the interaction of BCL10 and MALT1, BCL10 was found to be processed to a truncated form by MALT1. The findings lead us to the proposed studies thereafter.
2. identification of the phosphorylation site(s) of BCL10 and unraveling the myth of BCL10 phosphorylation.
Overexpression of BCL10 induced BCL10 phosphorylation, NF-B activation and the appearance of filamentous structure under microscope. BCL10L41R, a CARD mutated BCL10, lost all these BCL10-induced abilities. Neither N’BCL10GFP (1~114 a.a.) nor C’BCL10GFP (114~233 a.a.) was phosphorylated, indicating that both CARD and Serine/Threonine rich domain are required for BCL10 phosphorylation. Though not phosphorylated, N’BCL10GFP retained the ability to activate NF-B, suggesting that BCL10 phosphorylation was not essential for NF-B activation. The level of BCL10 phosphorylation was diminished by dominant-negative IκB through blocking NF-B activation.
Site directed mutagenesis was utilized to generate BCL10 mutants to map the phosphorylation sites on BCL10. In addition to the reported Serine 134,136,138,141 and 144, Serine 170 and 171 were also found to be major phosphorylation sites. Mutation with serine to alanine at these sites completely abolished the appearance of highly-molecular-weight phosphorylation isoforms. However, the un-phosphorylated BCL10 mutant retained its ability to activate NF-B and formed filamentous structure upon overexpression in 293T cells as wild type BCL10.
3. studying the nature of BCL10 cleavage
Arginine 228 was the reported cleavage site. As expected, BCL10R228G could not be cleaved by MALT1. However, in our assay system, deletion of Leucine225 abolished the nature of BCL10 being a substrate of MALT1. Site-directed mutagenesis was employed to investigate the role of amino acid residue Leu225 of BCL10 played as a substrate of MALT1. MALT1 failed to process BCL10L225A, BCL10L225G, BCL10L225Q, BCL10L225E mutants. But Mutants BCL10L225K and BCL10L225R could be processed by MALT1 as wild type BCL10. Interestingly, double mutants BCL10L225R,R228G retained its ability to be cleaved by MALT1. (The study has resulted in the publication of the following paper : J Biomed Sci. 2012; 19:85).
4. functional consequences of MALT1-mediated BCL10 cleavage
Truncated BCL10 induced NF-B activation and the appearance of filamentous structure under microscope, in analogy to wild type BCL10. Though phosphorylated, truncated BCL10 did display a different phosphorylation pattern as visualized by the different mobility of the phosphorylated products.
5. searching for physiological conditions that would trigger the cleavage of BCL10
Multiple cell lines including:Jurkat, Raji, Akata, NK, Raw264.7, THP-1, HeLa, AGS, and 293T were treated with stimulators, such as PMA/ionomycin, TNF-, LPS, Zymosan, IL-1, Helicobacter pylori, known to activate NF-B signaling. The levels of IB were examined as the indicator for NF-B activation. Cleavage of BCL10 or not could easily be monitored by denaturing polyacrylamide gel electrophoresis. Cleavage of BCL10 was observed only in cells(e.g. Jurkat, Raji, Akata, NK)of lymphoid origin in response to PMA/ionomycin treatment. Proteasome inhibitor, MG132, had no effect on the cleavage of BCL10. Bay 11-7085, IKK inhibitor, attenuated the level of BCL10 cleavage. Cytosolic calcium chelator, BAPTA, also downregulated the PMA/ionomycin-induced BCL10 cleavage. Taking together, IKK activity and Calcium might play certain roles in the cleavage of BCL10 in lymphoid cells.
6. generating constitutively-active paracaspase by fusion caspase-like domain of MALT1 with oligomierization motif from different molecules
Different fusion constructs (C106-2IgCLD,G-2IgCLD,and IAP2-2IgCLD)with oligomerization domain from caspase recruitment domain (CARD) of BCL10, dimerization domain of E. coli gyrase B and BIR domain of IAP2 were tested and compared for their abilities to activate NF-B or proteolytically process BCL10 substrate. Fusion construct C106-2IgCLD showed proteolytic activity but with minimum NF-B activation activity. G-2IgCLD showed potent NF-B activation activity but no proteolytic activity. IAP2-2IgCLD showed both potent NF-B activation and proteolytic activity. Fusion constructs with different oligomerization moiety showed apparently distinct properties. (Manuscript in preparation)