Glycan-Dependent Signaling and Regulation of Nuclear Tra

核运输对于维持转录因子、核蛋白和复制因子的水平和活性至关重要。我们发现Ca+2/钙调蛋白是核输入的激活剂，并建议在细胞激活过程中，Ca+2在核输入的调节中发挥作用。我们还展示了多功能凝集素钙网蛋白在核出口中的作用。我们认为，跨核孔的双向运输是由GTP和Ca+2控制的，从而为核运输和其他信号转导途径提供了协调调节。我们目前正在研究钙网蛋白可能不同地靶向内质网和核的机制，以及它在调节核输出中的作用。 翻译后修饰是基因调控的另一种方式。O-GlcNAc是丝氨酸/苏氨酸的胞内糖链修饰物，被认为通过与磷酸化竞争参与多种信号通路。我们试图了解O-GlcNAc依赖信号的生物学功能，并确定O-GlcNAc代谢改变是否与糖尿病和神经退行性变等人类疾病有关。O-连接的GlcNAc转移酶(OGT)和O-GlcNAcase分别催化O-GlcNAc的加成和去除。O-GlcNAc修饰的细胞靶标主要是核孔复合体和转录复合体。NPC通过核膜调节大分子的运输，在后生动物中，NPC被O-GlcNAc广泛修饰。转录复合体也是O-GlcNAc修饰的关键靶点。重要的是，与组蛋白脱乙酰酶一样，OGT被招募到Sin3a转录-抑制复合体中。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。O-GlcNAc被转移到UDP-GlcNAc的蛋白质中，UDP-GlcNAc是一种糖核苷酸，其水平受氨基己糖生物合成途径(HBP)的调节，起着细胞传感器的作用。营养的可获得性。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L的肌肉中，通过高血压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。研究O-GlcNAc代谢酶的结构、靶向和调节是我们的主要重点。我们在大肠杆菌中表达了具有全功能的OGT和O-GlcNAcase。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。对于O-GlcNAcase，我们发现只有含有GCN5样乙酰基转移酶结构域的长异构体保持了催化活性。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。线粒体和核异构体OGT的不同定位表明，它们在细胞内分别在细胞凋亡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向。当OGT过表达时，糖依赖信号被诱导升高，从而导致细胞程序性死亡。转基因过表达OGT亚型在肌肉和脂肪中诱导小鼠胰岛素抵抗和高瘦素血症。这些数据证明了OGT在胰岛素和瘦素信号级联中的核心作用。这些发现表明，糖依赖的信号在营养感知和II型糖尿病的发病机制中具有更普遍的作用。利用反向遗传学、基因敲除和其他转基因模型，我们目前正在探索O-GlcNAc代谢酶在信号转导和糖尿病发病机制中的作用。我们还鉴定了一株缺乏OGT活性的线虫活菌株，目前我们正在研究该缺失对线虫发育和生理的影响。

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.