Genomic structure and subcellular localization of MAL, a human T-cell-specific proteolipid protein.

ABSTRACT Bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2) belong to the same subfamily of oncoviruses. Defective HTLV-1 proviral genomes have been found in more than half of all patients with adult T-cell leukemia examined. We have characterized the genomic structure of integrated BLV proviruses in peripheral blood lymphocytes and tumor tissue taken from animals with lymphomas at various stages. Genomic Southern hybridization with SacI, which generates two major fragments of BLV proviral DNA, yielded only bands that corresponded to a full-size provirus in all of 23 cattle at the lymphoma stage and in 7 BLV-infected but healthy cattle. Long PCR with primers located in long terminal repeats clearly demonstrated that almost the complete provirus was retained in all of 27 cattle with lymphomas and in 19 infected but healthy cattle. However, in addition to a PCR product that corresponded to a full-size provirus, a fragment shorter than that of the complete virus was produced in only one of the 27 animals with lymphomas. Moreover, when we performed conventional PCR with a variety of primers that spanned the entire BLV genome to detect even small defects, PCR products were produced that specifically covered the entire BLV genome in all of the 40 BLV-infected cattle tested. Therefore, it appears that at least one copy of the full-length BLV proviral genome was maintained in each animal throughout the course of the disease and, in addition, that either large or small deletions of proviral genomes may be very rare events in BLV-infected cattle.

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The Degradation and Subcellular Localization of an Oncogenic protein complex in T-cell Leukemia

Proceedings of IMPRS ◽

10.18060/22824 ◽

2018 ◽

Vol 1 (1) ◽

Author(s):

Chiagozie Nwosu ◽

Michael Christy ◽

Justin Layer ◽

Utpal Davé

Keyword(s):

T Cell ◽

Subcellular Localization ◽

Protein Stability ◽

Half Life ◽

Lymphoblastic Leukemia ◽

Lim Domain ◽

Helix Loop Helix ◽

Human T Cell ◽

Protein Components ◽

Oncogenic Protein

Background and Hypothesis: LIM domain Only-2 (LMO2) is a key oncogenic driver of human T-cell acute lymphoblastic leukemia. LMO2 functions as part of a large multisubunit complex that regulates gene expression. LMO2 itself does not bind DNA but it does bind class II basic helix loop helix transcription factors, TAL1 or LYL1, which are also part of the LMO2-associated complex. We recently identified LIM domain binding protein 1 (LDB1) as an obligate LMO2 partner that is required for LMO2 protein stability in T-ALL. We believe the interaction between LDB1 and LMO2 is crucial to understanding the pathogenesis of T-ALL and is a potential therapeutic target for this aggressive leukemia. We hypothesize that in the absence of LDB1, LMO2 is rapidly degraded. Nonetheless, the mechanisms for the localization and degradation of LMO2 and its partners have not been fully explored. Experimental Design or Project Methods: In this study, we analyzed the first order decay of the LMO2-associated complex including LMO2, SSBP2, SSBP3, TAL1, LYL1, and LDB1. We analyzed the subcellular localization of these same proteins distinguishing between the nucleus and the cytoplasm. Our studies were enabled by cloning the HALO tag to the NH2-terminus of these proteins. Fluorescent small molecules such as HALO ligand, R110, were used for intracellular labeling followed by pulse chase analysis via flow cytometry for half-life and imaging by confocal microscopy. Results: We observed a hierarchy of protein stability: LDB1 had the longest half-life, followed by SSBP3, SSBP2, Tal1, LMO2 and Lyl1had the shortest half-lives. Co-expression of LDB1 conferred enhanced staility upon all protein components. All of the protein components studied showed a predominantly nuclear distribution with some in the cytoplasm. Conclusion and Potential Impact: The prolonged protein stability of LDB1 allows it to confer enhanced stability to LMO2 and bHLH proteins TAL1 and LYL1. Thus, targeting the assembly of the multisubunit LMO2 complex.

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