Glycan-Dependent Signaling and Regulation of Nuclear Tra

核转录的聚糖依赖性信号传导和调节

• 批准号: 6810570
• 负责人: John A. Hanover
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6810570
• 关键词: apoptosis; biological signal transduction; calcium flux; calmodulin; cell nucleus; enzyme activity; gene expression; gene targeting; genetic transcription; genetically modified animals; guanosine triphosphate; intracellular transport; neural degeneration; noninsulin dependent diabetes mellitus; nuclear membrane; protein transport; transcription factor

Nuclear transport is critical for the maintenance of the levels and activities of transcription factors, nuclear kinases, and replication factors. We identified Ca+2/calmodulin as an activator of nuclear import and suggested a role for Ca+2 in the regulation of nuclear import during cell activation. We also demonstrated a role for the multifunctional lectin calreticulin in nuclear export. We suggest that bi-directional transport across the nuclear pore is controlled by GTP and Ca+2, thus providing coordinate regulation of nuclear transport and other signal transduction pathways. We are currently examining the mechanism by which Calreticulin may be differentially targeted to the endoplasmic reticulum and the nucleus and its role in modulating nuclear export. Posttranslational modifications are another means by which gene regulation may be regulated. O-GlcNAc is an intracellular glycan modification of Ser/Thr proposed to participate in diverse signaling pathways, via competition with phosphorylation. We seek to understand the biological functions of O-GlcNAc-dependent signaling and to determine whether altered O-GlcNAc metabolism contributes to human diseases such as diabetes mellitus and neurodegeneration. Addition and removal of O-GlcNAc are catalyzed by O-linked GlcNAc transferase (OGT) and O-GlcNAcase, respectively. The best-characterized cellular targets modified by O-GlcNAc are nuclear pore complexes (NPC) and transcription complexes. The NPC mediates macromolecular traffic across the nuclear membrane, and in metazoans, the NPC is extensively modified by O-GlcNAc. Transcription complexes are also key targets for O-GlcNAc modification. Importantly, as with histone deacetylases, OGT is recruited to Sin3a transcription-repression complexes. 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. 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 skeleta l 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 which 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. Examining the structure, targeting, and regulation of the enzymes of O-GlcNAc metabolism is our principal focus. We expressed fully functional OGT and O-GlcNAcase in E. coli. 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. For O-GlcNAcase, we found that only the long isoform containing a GCN5-like acetyltransferase domain retained catalytic activity. 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 and transcriptional repression respectively. O-GlcNAcase isoforms are also differentially targeted in cells. Elevated Glycan-dependent signaling was induced upon overexpression of OGT and this induced programmed cell death. Transgenic overexpression of an isoform of OGT in muscle and fat Induced Insulin resistance and Hyperleptinemia in mice. These data demonstrate a central role for OGT in the insulin and leptin-signaling cascades. The findings suggest a more general role for glycan-dependent signaling in nutrient sensing and the pathogenesis of Type II diabetes. Using reverse genetics, knockout, and other transgenic models we are currently exploring the role of the enzymes of O-GlcNAc metabolism in signal transduction and the pathogenesis of diabetes mellitus. We have also identified a viable Caenorhabditis elegans strain lacking OGT activity and we are currently examining the effects of the deletion on nematode development and physiology.

核转运对于维持转录因子、核激酶和复制因子的水平和活性至关重要。我们确定了Ca+2/钙调素作为核输入的激活剂，并建议在细胞活化过程中的调节核输入的Ca+2的作用。我们还证明了多功能凝集素钙网蛋白在核输出中的作用。我们认为，通过核孔的双向运输是由GTP和Ca+2控制的，从而提供协调调节核运输和其他信号转导途径。目前，我们正在研究钙网蛋白的机制，可能是不同的针对内质网和细胞核及其在调节核输出的作用。 翻译后修饰是基因调控的另一种方式。O-GlcNAc是Ser/Thr的细胞内聚糖修饰，被认为通过与磷酸化竞争参与多种信号传导途径。我们试图了解O-GlcNAc依赖性信号传导的生物学功能，并确定O-GlcNAc代谢的改变是否有助于人类疾病，如糖尿病和神经变性。O-GlcNAc的添加和去除分别由O-连接的GlcNAc转移酶（OGT）和O-GlcNAc酶催化。O-GlcNAc修饰的最具特征的细胞靶标是核孔复合物（NPC）和转录复合物。NPC介导跨核膜的大分子运输，并且在后生动物中，NPC被O-GlcNAc广泛修饰。转录复合物也是O-GlcNAc修饰的关键靶标。重要的是，与组蛋白脱乙酰酶一样，OGT被募集到Sin 3a转录抑制复合物中。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。O-GlcNAc从UDP-GlcNAc转移到蛋白质中，UDP-GlcNAc是一种糖核苷酸，其水平受充当细胞？传感器？的养分供应。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。在胸肌中，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的成纤维细胞表现出增殖、增殖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。研究O-GlcNAc代谢酶的结构、靶向和调节是我们的主要关注点。我们在大肠杆菌中表达了功能完整的OGT和O-GlcNAc酶。杆菌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。对于O-GlcNAcase，我们发现只有含有GCN 5样乙酰转移酶结构域的长同种型保留催化活性。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。在OGT过表达时诱导聚糖依赖性信号传导升高，这诱导了程序性细胞死亡。肌肉和脂肪中OGT亚型的转基因过表达诱导小鼠的胰岛素抵抗和高瘦素血症。这些数据表明OGT在胰岛素和瘦素信号级联中的核心作用。这些发现表明，聚糖依赖性信号在营养感知和II型糖尿病发病机制中具有更普遍的作用。我们目前正在利用反向遗传学、基因敲除和其他转基因模型探索O-GlcNAc代谢酶在信号转导和糖尿病发病机制中的作用。我们还确定了一个可行的秀丽隐杆线虫菌株缺乏OGT活性，我们目前正在研究的删除对线虫发育和生理的影响。