Enzymes of O-GlcNAc cycling linked to type-2 diabetes and neurodegeneration

细胞核和细胞质中O-GlcNAc的添加和去除的动态循环介导己糖胺信号传导途径中的最后一步。 这种修饰的靶点是核孔复合物、转录复合物、蛋白酶体和信号激酶。基于O-GlcNAc修饰的靶点，我们提出O-GlcNAc代谢的酶调节核转运、转录、细胞生长和凋亡以响应营养的可用性。研究O-GlcNAc代谢酶的结构、靶向和调节是我们的主要关注点。O-GlcNAc从UDP-GlcNAc转移到蛋白质中，UDP-GlcNAc是一种糖核苷酸，其水平受己糖胺生物合成途径（HBP）调节，作为营养物质可用性的细胞传感器。通过整合这些信号，HBP调节包括瘦素在内的许多基因产物的表达。在骨骼肌中，通过HBP的通量与胰岛素抵抗的程度相关。HBP还与调节细胞增殖和凋亡的途径有关;不能乙酰化UDP-GlcNAc的成纤维细胞表现出增殖、凋亡和对凋亡刺激的抗性的缺陷。因此，通过产生UDP-GlcNAc，HBP可以被视为营养物传感信号传导途径。我们试图确定O-GlcNAc如何参与这种信号级联。 我们正在检验O-GlcNAc代谢酶的差异靶向异构体介导这种聚糖依赖性信号传导途径的假设。通过响应营养水平，该途径调节基因表达，细胞生长和程序性细胞死亡。我们在E.杆菌我们最近解决了OGT的超螺旋TPR（tetratricopeptide repeat）结构域的结构，该结构域介导靶蛋白的识别，并显示出与importin alpha的结构相似性。与作为信号分子的作用一致，我们表明OGT修饰糖原合成酶激酶-3和酪蛋白激酶，这两种酶调节糖原合成。O-GlcNAc循环的大部分影响通过转录的变化发生。重要的是，组蛋白脱乙酰酶（HDAC）和OGT都被募集到Sin 3a转录抑制复合物中。O-GlcNAcase的一种同种型的C-末端已被证明是组蛋白乙酰转移酶（HAT）。因此，O-GlcNAc似乎是组蛋白重塑复合物的动态参与者。 我们目前正在测试这一假设，通过干扰O-GlcNAc循环，并使用芯片上芯片，高通量测序和表达阵列技术检查对染色质的后续影响。 我们还关注了O-GlcNAc循环酶的催化功能。我们证明了O-GlcNAc酶的两种亚型都是调节细胞O-GlcNAc水平的活性酶。OGT和O-GlcNAc酶的突变分析使我们能够定义催化结构域。我们发现OGT亚型靶向细胞核和线粒体。OGT的线粒体和核异构体的差异定位表明，它们分别在细胞凋亡、线粒体运动和转录抑制中发挥独特的细胞内功能。O-GlcNAc酶同种型在细胞中也有不同的靶向;一种同种型是细胞核，而另一种在脂质储存的细胞位点积累。正在使用合成和天然产物方法开发O-GlcNAc代谢酶的小分子抑制剂和底物。我们已经提出，这种细胞内的聚糖修饰的丝氨酸/苏氨酸参与了不同的信号通路的方式类似于蛋白质磷酸化。 在合作研究中，我们已经表明，人类OGT基因是受X染色体印记，似乎在冠状动脉疾病的易感性中发挥关键作用。 我们还采用化学生物学方法来研究O-GlcNAc循环。 我们首先开发了用于检测O-GlcNAc添加的化学方法。 该方法用于高通量筛选以鉴定O-GlcNAc循环的抑制剂。 我们还开发了许多O-GlcNAc酶特异性荧光底物和抑制剂，这将有助于剖析与2型糖尿病、肥胖和神经变性有关的己糖胺信号通路。这些试剂将有助于解剖的营养感应己糖胺信号通路。 O-GlcNAc循环的酶可能在与代谢疾病相关的信号传导和表观遗传景观的变化中起关键作用。

A dynamic cycle of addition and removal of O-GlcNAc in the nucleus and cytoplasm mediates a final step in the hexosamine signaling pathway. The targets of this modification are nuclear pore complexes, transcription complexes, proteasomes and signaling kinases. Based on the targets modified by O-GlcNAc, we proposed that the enzymes of O-GlcNAc metabolism modulate nuclear transport, transcription, cell growth, and apoptosis in response to nutrient availability. Examining the structure, targeting, and regulation of the enzymes of O-GlcNAc metabolism is our principal focus. O-GlcNAc is transferred to proteins from UDP-GlcNAc, a sugar nucleotide whose levels are regulated by the hexosamine biosynthetic pathway (HBP) acting as a cellular sensor of nutrient availability. By integrating these signals, the HBP regulates expression of a number of gene products that include leptin. In skeletal muscle, flux through the HBP correlates with the degree of insulin resistance. The HBP is also linked to pathways regulating cell proliferation and apoptosis; fibroblasts that cannot acetylate UDP-GlcNAc exhibit defects in proliferation, adhesiveness and resistance to apoptotic stimuli. Thus, by generating UDP-GlcNAc, the HBP may be viewed as a nutrient-sensing signaling pathway. We seek to determine how O-GlcNAc participates in this signaling cascade. We are testing the hypothesis that differentially targeted isoforms of the enzymes of O-GlcNAc metabolism mediate this glycan-dependent signaling pathway. By responding to nutrient levels, this pathway modulates gene expression, cell growth and programmed cell death. We expressed fully functional OGT and O-GlcNAcase isoforms in E. coli. We recently solved the structure of the superhelical TPR (tetratricopeptide repeat) domain of OGT that mediates the recognition of target proteins and showed that exhibits structural similarities to importin alpha. Consistent with a role as a signaling molecule, we showed that OGT modifies glycogen synthase kinase-3 and casein kinase, two enzymes regulating glycogen synthesis. Much of the impact of O-GlcNAc cycling occurs through changes in transcription. Importantly, both histone deacetylases (HDAC) and OGT are recruited to Sin3a transcription-repression complexes. The C-terminus of one isoform of the O-GlcNAcase has been shown to be a histone acetyltransferase (HAT). Therefore, O-GlcNAc appears to be a dynamic participant in histone remodeling complexes. We are currently testing this hypothesis by interfering with O-GlcNAc cycling and examining the subsequent impact on chromatin using CHIP-on-Chip, high throughput sequencing and expression array technologies. We have also focused on the catalytic functions of the enzymes of O-GlcNAc cycling. We demonstrated that both isoforms of O-GlcNAcase are active enzymes modulating cellular O-GlcNAc levels. Mutational analysis of OGT and O-GlcNAcase allowed us to define catalytic domains. We showed that OGT isoforms are targeted to both nucleus and mitochondria. The differential localization of mitochondrial and nuclear isoforms of OGT argues that they perform unique intracellular functions in apoptosis, mitochondrial movement and transcriptional repression respectively. O-GlcNAcase isoforms are also differentially targeted in cells; one isoform is nuclear while another accumulates at cellular sites of lipid storage. Small molecule inhibitors and substrates for the enzymes of O-GlcNAc metabolism are under development using both synthetic and natural product approaches. We have proposed that this intracellular glycan modification of Ser/Thr participates in diverse signaling pathways in a manner analogous to protein phosphorylation. In collaborative studies, we have shown that the human OGT gene is subject to X-chromosome imprinting and appears to play a key role in the susceptibility to coronary artery disease. We also have taken Chemical Biology approaches to examine O-GlcNAc cycling. We first developed a chemical method for detecting O-GlcNAc addition. This method is being used for high throughput screening to identify inhibitors of O-GlcNAc cycling. We have also developed a number of O-GlcNAcase-specific fluorogenic substates and inhibitors that will facilitate dissection of the hexosamine signaling pathway implicated in Type-2 diabetes, obesity and neurodegeneration. These reagents will facilitate the dissection of the nutrient-sensing hexosamine signaling pathway. The enzymes of O-GlcNAc cycling may play a key role in the changes in signaling and epigenetic landscape associated with metabolic disease.