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

O-GlcNAc在细胞核和细胞质中的添加和移除的动态循环介导了氨基己糖信号通路的最后一步。这种修饰的靶点是核孔复合体、转录复合体、蛋白酶体和信号转导酶。基于O-GlcNAc修饰的靶点，我们认为O-GlcNAc代谢的酶调控核运输、转录、细胞生长和细胞凋亡，以响应营养的供应。研究O-GlcNAc代谢酶的结构、靶向和调节是我们的主要重点。O-GlcNAc被转移到UDP-GlcNAc的蛋白质中，UDP-GlcNAc是一种糖核苷酸，其水平受氨基己糖胺生物合成途径(HBP)的调节，充当营养可获得性的细胞传感器。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的流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的成纤维细胞在增殖、粘附性和对凋亡刺激的抵抗力方面存在缺陷。因此，通过产生UDP-GlcNAc，HBP可以被视为一条营养感知信号通路。我们试图确定O-GlcNAc如何参与这一信号级联。 我们正在验证这样的假设，即O-GlcNAc代谢酶的不同靶向亚型介导了这一依赖于糖的信号通路。通过对营养水平的反应，这一途径调节基因表达、细胞生长和细胞程序性死亡。我们在大肠杆菌中表达了具有全功能的OGT和O-GlcNAcase亚型。我们最近解决了OGT的超螺旋TPR(四肽重复)结构域的结构，该结构介导了靶蛋白的识别，并显示出与Importinα的结构相似。与作为信号分子的作用一致，我们发现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循环的大部分影响是通过转录的变化来实现的。重要的是，组蛋白脱乙酰酶(HDAC)和OGT都被招募到Sin3a转录-抑制复合体中。O-GlcNAcase的一个异构体的C末端是一个组蛋白乙酰转移酶(HAT)。因此，O-GlcNAc似乎是组蛋白重塑复合体的动态参与者。我们目前正在通过干扰O-GlcNAc循环来验证这一假设，并使用芯片上芯片、高通量测序和表达阵列技术来检查随后对染色质的影响。 我们还重点研究了O-GlcNAc循环中酶的催化作用。我们证明了O-GlcNAcase的两种异构体都是调节细胞O-GlcNAc水平的活性酶。对OGT和O-GlcNAcase的突变分析使我们能够确定催化结构域。我们发现OGT亚型针对细胞核和线粒体。OGT线粒体和核异构体的不同定位表明，它们在细胞内分别在细胞凋亡、线粒体运动和转录抑制中发挥独特的功能。O-GlcNAcase亚型在细胞中也有不同的靶点；一个亚型是核的，而另一个亚型聚集在储存脂质的细胞部位。用于O-GlcNAc代谢酶的小分子抑制剂和底物正在开发中，使用合成和天然产品的方法。我们已经提出，Ser/Thr的这种细胞内糖链修饰以类似于蛋白质磷酸化的方式参与了不同的信号通路。在合作研究中，我们已经证明人类OGT基因受到X染色体印记的影响，并似乎在冠状动脉疾病的易感性中发挥关键作用。 我们还采用了化学生物学的方法来研究O-GlcNAc循环。我们首先建立了一种检测O-GlcNAc加成的化学方法。该方法正被用于高通量筛选，以确定O-GlcNAc循环的抑制剂。我们还开发了一些O-GlcNAcase特异性的荧光亚态和抑制剂，这将有助于剖析与2型糖尿病、肥胖症和神经退化有关的氨基己糖信号通路。这些试剂将有助于对营养敏感的氨基己糖信号通路的剖析。 O-GlcNAc循环的酶可能在与代谢性疾病相关的信号和表观遗传格局的变化中发挥关键作用。

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.