Identification of novel regulators of mitotic exit in Saccharomyces cerevisiae

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2006.Includes bibliographical references.The division of a eukaryotic cell into two daughter cells is controlled by cyclin dependent kinase (CDK). Entry into mitosis is promoted by the activity of CDK complexed with mitotic cyclins. Upon faithful segregation of a full complement of DNA between each daughter cell, exit from mitosis proceeds. In order for cells to exit from mitosis and enter into G 1, mitotic CDKs must be inactivated. In Saccharomyces cerevisiae, mitotic exit is regulated by two signaling networks, the mitotic exit network (MEN) and the Cdc14 early anaphase release (FEAR) network. In this budding yeast, coordination of nuclear migration and mitotic exit is critical to prevent aneuploidy. A surveillance mechanism known as the spindle position checkpoint ensures that exit from mitosis only occurs when the anaphase nucleus is positioned along the mother - bud axis. The work presented here describes two screens that have isolated novel regulators of mitotic exit. A model for the regulation of mitotic exit by the gene KIN4 is proposed. This work identifies the protein kinase Kin4 as a component of the spindle position checkpoint.Katharine E. D'Aquino.Ph.D

Unique pharmacology of a novel allosteric agonist/sensitizer insulin receptor monoclonal antibody

Objective: Insulin resistance is a key feature of Type 2 Diabetes (T2D), and improving insulin sensitivity is important for disease management. Allosteric modulation of the insulin receptor (IR) with monoclonal antibodies (mAbs) can enhance insulin sensitivity and restore glycemic control in animal models of T2D. Methods: A novel human mAb, IRAB-A, was identified by phage screening using competition binding and surface plasmon resonance assays with the IR extracellular domain. Cell based assays demonstrated agonist and sensitizer effects of IRAB-A on IR and Akt phosphorylation, as well as glucose uptake. Lean and diet-induced obese mice were used to characterize single-dose in vivo pharmacological effects of IRAB-A; multiple-dose IRAB-A effects were tested in obese mice. Results: In vitro studies indicate that IRAB-A exhibits sensitizer and agonist properties distinct from insulin on the IR and is translated to downstream signaling and function; IRAB-A bound specifically and allosterically to the IR and stabilized insulin binding. A single dose of IRAB-A given to lean mice rapidly reduced fed blood glucose for approximately 2 weeks, with concomitant reduced insulin levels suggesting improved insulin sensitivity. Phosphorylated IR (pIR) from skeletal muscle and liver were increased by IRAB-A; however, phosphorylated Akt (pAkt) levels were only elevated in skeletal muscle and not liver vs. control; immunochemistry analysis (IHC) confirmed the long-lived persistence of IRAB-A in skeletal muscle and liver. Studies in diet-induced obese (DIO) mice with IRAB-A reduced fed blood glucose and insulinemia yet impaired glucose tolerance and led to protracted insulinemia during a meal challenge. Conclusion: Collectively, the data suggest IRAB-A acts allosterically on the insulin receptor acting non-competitively with insulin to both activate the receptor and enhance insulin signaling. While IRAB-A produced a decrease in blood glucose in lean mice, the data in DIO mice indicated an exacerbation of insulin resistance; these data were unexpected and suggested the interplay of complex unknown pharmacology. Taken together, this work suggests that IRAB-A may be an important tool to explore insulin receptor signaling and pharmacology. Keywords: Insulin, Insulin receptor, Positive allosteric modulator, Monoclonal antibody, Diabete

Insulin Receptor Substrate-2 in β-Cells Decreases Diabetes in Nonobese Diabetic Mice

Insulin receptor substrate-2 (Irs2) integrates insulin-like signals with glucose and cAMP agonists to regulate β-cell growth, function, and survival. This study investigated whether increased Irs2 concentration in β-cells could reduce β-cell destruction and the incidence of type 1 diabetes in nonobese diabetic (NOD) mice. NOD mice were intercrossed with C57BL/6 mice overexpressing Irs2 specifically in β-cells to create NODIrs2 mice. After backcrossing NODIrs2 mice for 12 generations, glucose homeostasis and diabetes incidence were compared against NOD littermates. Compared with 12-wk-old NOD mice, the progression of severe insulitis was reduced and islet mass was increased in NODIrs2 mice. Moreover, the risk of diabetes decreased 50% in NODIrs2 mice until the experiment was terminated at 40 wk of age. Nondiabetic NODIrs2 mice displayed better glucose tolerance than nondiabetic NOD mice throughout the duration of the study and up to the age of 18 months. The effect of Irs2 to increase islet mass and improve glucose tolerance raised the possibility that NODIrs2 mice might have an increased capacity to respond to anti-CD3 antibody, which can induce remission of overt diabetes in some NOD mice. Anti-CD3 antibody injections restored glucose tolerance in newly diabetic NOD and NODIrs2 mice; however, anti-CD3-treated NODIrs2 mice were less likely than NOD mice to relapse during the experimental period because they displayed 10-fold greater β-cell mass and mitogenesis. In conclusion, increased Irs2 attenuated the progression of β-cell destruction, promoted β-cell mitogenesis, and reduced diabetes incidence in NODIrs2 mice