Investigations of Golgi Enzymes

性状（由申请方提供）：细胞表面聚糖是细胞-细胞和细胞-基质相互作用的主要决定因素。它们的结构反映了在高尔基体隔室内的装配线中起作用的糖基转移酶和磺基转移酶的表达。该项目的主要目标是开发化学工具，用于研究高尔基体酶及其在细胞上产生的聚糖的功能。最后一个授权期集中在一个新发现的GlcNAc-6-磺基转移酶家族。目标是（1）开发磺基转移酶的小分子抑制剂作为生物学研究的工具和药物发现的线索，（2）鉴定参与底物结合和催化的残基，以及（3）确定优选的细胞底物作为阐明生物学功能的一个步骤。这项工作的一个令人兴奋的发现是，体内每种酶的底物偏好在很大程度上取决于其在高尔基体中的分布。因此，高尔基体定位被确定为生物学功能的主要决定因素。下一个授予期的具体目标是从这一发现中建立的。下一个授权期的主要目标是开发一种用小分子调节高尔基酶活性的方法，以靶向它们对高尔基定位的共同要求。所提出的策略是基于化学二聚化诱导的组装酶的模块化催化和定位结构域。该方法用岩藻糖基转移酶7（FucT 7）和GlcNAc-6-磺基转移酶GST-2和GST-3验证，使用雷帕霉素/FRB/FKBP系统进行诱导结构域组装。下一个授权期的具体目标在三个方向上扩展了这一发现。第一个目的是更详细地研究FucT 7系统，以定义影响重构结构域的细胞活性的那些参数。我们的目标是优化目前的FucT 7系统应用于肿瘤细胞转移的研究，并在转基因小鼠中使用。第二个目的是将该方法应用于其他高尔基体酶，选择其多样性的底物和功能。第三个也是最后一个目的是确定化学二聚体是否可以用于调节两个糖基转移酶之间的关联。这种关联被认为对糖脂生物合成途径的效率很重要。调节糖基转移酶缔合的能力将提供控制细胞上糖脂表达的手段。

DESCRIPTION (provided by applicant): Cell surface glycans are major determinants of cell-cell and cell-matrix interactions. Their structures reflect the expression of glycosyltransferases and sulfotransferases that act in an assembly line within the Golgi compartment. The broad objective of this project is to develop chemical tools for studying Golgi enzymes and the functions of the glycans they produce on cells. The last granting period focused on a newly discovered family of GlcNAc-6-sulfotransferases. The goals were to (1) develop small molecule inhibitors of the sulfotransferases as tools for biological studies and leads for drug discovery, (2) identify residues involved in substrate binding and catalysis, and (3) determine the preferred cellular substrates as a step toward elucidating biological function. An exciting discovery from this work was that the substrate preference of each enzyme in vivo is governed largely by its distribution among the Golgi cisternae. Thus, Golgi localization was identified as a major determinant of biological function. The Specific Aims of the next granting period build from this discovery. The major objective of the next granting period is to develop an approach for modulating Golgi enzyme activity with small molecules that target their common requirement of Golgi localization. The proposed strategy is based on the chemical dimerizer-induced assembly of the enzymes' modular catalytic and localization domains. The approach was validated with fucosyltransferase 7 (FucT7) and the GlcNAc-6- sulfotransferases GST-2 and GST-3, using the rapamycin/FRB/FKBP system for inducible domain assembly. The Specific Aims of the next granting period expand upon this discovery in three directions. The first Aim is to investigate the FucT7 system in more detail in order to define those parameters that affect the cellular activity of the reconstituted domains. The goal is to optimize the current FucT7 system for application to studies of tumor cell metastasis and for use in transgenic mice. The second Aim is to apply the approach to other Golgi enzymes, chosen for their diversity of substrates and functions. The third and final Aim is to determine whether chemical dimerizers can be used to modulate associations between two glycosyltransferases. Such associations are thought to be important for the efficiency of glycolipid biosynthetic pathways. The ability to modulate glycosyltransferase associations will provide means to control glycolipid expression on cells.