Targeting and Internalization Mechanism of LFA-1

LFA-1的靶向和内化机制

• 批准号: 7209818
• 负责人: TERUNA J. SIAHAAN
• 金额: $ 26.98万
• 依托单位: UNIVERSITY OF KANSAS LAWRENCE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7209818
• 关键词: Apoptosis; Arthritis; Autoimmune Diseases; Binding; Cell Adhesion Inhibition; Cell Death; Cell surface; Cells; Collagen Arthritis; Cultured Cells; Cytoplasm; Cytoplasmic Tail; Cytotoxic agent; Dose; Drug Delivery Systems; Endosomes; Epithelial Cells; Evaluation; Figs - dietary; Fluorescein; Fluoresceins; Folic Acid; Goals; Human; In Vitro; Integrins; Intercellular adhesion molecule 1; Isothiocyanates; Kinetics; Label; Leukocytes; Link; Localized; Lymphocyte Function-Associated Antigen-1; Measures; Mediating; Membrane; Methods; Methotrexate; Modeling; Mus; Peptides; Pharmaceutical Preparations; Process; Process Measure; Production; Proteins; Recycling; Research Design; Research Personnel; Resistance; Rheumatoid Arthritis; Role; SLC19A1 gene; Signal Transduction; Surface; System; T-Cell Activation; T-Cell Leukemia; T-Lymphocyte; Toxic effect; crosslink; cytohesin-1; cytokine; cytotoxicity; folate-binding protein; in vivo Model; leukemia; mouse model; programs; receptor; response; uptake

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to understand the mechanism of internalization of integrins and their utility for targeting drugs to specific cells. The short-term objective of this proposal is to study the mechanism of internalization of LFA-1 on T cells and to utilize this mechanism for targeting drugs to leukocytes. The central hypothesis is that an ICAM-1-derived peptide (cIBR) can bind to and be internalized by LFA-1. Therefore, a cytotoxic drug (methotrexate, MTX) will be linked with cIBR peptide to give MTX-cIBR conjugate; this conjugate will have dual functions: (a) to inhibit T-cell activation via inhibition of the cell adhesion signal and (b) to induce T-cell death by internalizing MTX. The MTX-cIBR conjugate has selective toxicity for LFA-1-expressing T cells but not LFA-1-deficient KB epithelial cells. MTXcIBR conjugate suppressed rheumatoid arthritis in the collagen-induced arthritis (CIA) mouse model better than MTX alone. Therefore, the first objective is to evaluate the mechanism of suppression of arthritis by MTX-cIBR conjugate (Specific Aim 1). The mechanism of MTX-cIBR conjugate in altering the activation of T cells from Th1 to Th2 response in the CIA mouse model will be elucidated. In addition, the long-term kinetic effects of MTX-cIBR on the T-cell commitment in the CIA mouse model will be determined. Secondly, the internalization of MTX-cIBR or FITC-cIBR conjugates will be released by LFA-1 in the cytoplasm; alternatively, it could (a) remain with the endosome, (b) be recycled to the cell surface, or (c) be targeted to other membrane compartments. Therefore, the internalization process and localization of these conjugates will be determined (Specific Aim 2). Thirdly, the internalization of MTX-cIBR may also be via reduced folate carrier (RFC) or membrane folate binding protein (mFBP). In Specific Aim 3, MTX-cIBR will be compared using wild type and RFC-deficient mouse leukemia T cells. In addition, this conjugate will be compared with MTX in mFBP over-expressing leukemia mouse cells in the presence and absence of folic acid. Finally, the exact mechanism of the LFA-1 internalization and the identity of other proteins that are involved in this process are not well understood. In Specific Aim 4, cIBR peptide will be modified and cross-linked to LFA-1 on activated T cells; the crosslink will be isolated along with proteins that interact with LFA-1. These isolated proteins will be identified.

描述（由申请人提供）：该提案的长期目标是了解整合素的内化机制及其将药物靶向特定细胞的效用。该提案的短期目标是研究LFA-1在T细胞上的内化机制，并利用该机制将药物靶向白细胞。核心假设是 ICAM-1 衍生肽 (cIBR) 可以与 LFA-1 结合并被 LFA-1 内化。因此，细胞毒性药物（甲氨蝶呤，MTX）将与cIBR肽连接，得到MTX-cIBR缀合物；该缀合物具有双重功能：(a) 通过抑制细胞粘附信号来抑制 T 细胞活化，(b) 通过内化 MTX 来诱导 T 细胞死亡。 MTX-cIBR 缀合物对表达 LFA-1 的 T 细胞具有选择性毒性，但对 LFA-1 缺陷的 KB 上皮细胞没有选择性毒性。 MTXcIBR 缀合物在胶原诱导关节炎 (CIA) 小鼠模型中比单独使用 MTX 更好地抑制类风湿性关节炎。因此，第一个目标是评估 MTX-cIBR 缀合物抑制关节炎的机制（具体目标 1）。将阐明 MTX-cIBR 缀合物在 CIA 小鼠模型中将 T 细胞活化从 Th1 反应改变为 Th2 反应的机制。此外，还将确定 MTX-cIBR 对 CIA 小鼠模型中 T 细胞定向的长期动力学影响。其次，MTX-cIBR或FITC-cIBR缀合物的内化会被细胞质中的LFA-1释放；或者，它可以（a）保留在内体中，（b）再循环到细胞表面，或（c）靶向其他膜区室。因此，将确定这些缀合物的内化过程和定位（具体目标 2）。第三，MTX-cIBR的内化也可能是通过还原叶酸载体(RFC)或膜叶酸结合蛋白(mFBP)。在具体目标 3 中，将使用野生型和 RFC 缺陷型小鼠白血病 T 细胞对 MTX-cIBR 进行比较。此外，在叶酸存在和不存在的情况下，该缀合物将与过表达 mFBP 的白血病小鼠细胞中的 MTX 进行比较。最后，LFA-1 内化的确切机制以及参与此过程的其他蛋白质的身份尚不清楚。在具体目标 4 中，cIBR 肽将被修饰并与激活的 T 细胞上的 LFA-1 交联；交联将与与 LFA-1 相互作用的蛋白质一起被分离。这些分离的蛋白质将被鉴定。