The Molecular Basis of Lysine Acetylation: Addition, Removal, and Recognition

Acetyltransferases and deacetylases catalyze the addition and removal, respectively, of acetyl groups to the epsilon-amino group of protein lysine residues. This modification can affect the function of a protein through several means, including the recruitment of specific binding partners called acetyl-lysine readers. Acetyltransferases, deacetylases, and acetyl-lysine readers have emerged as crucial regulators of biological processes and prominent targets for the treatment of human disease. This work describes a combination of structural, biochemical, biophysical, cell-biological, and organismal studies undertaken on a set of proteins that cumulatively include all steps of the acetylation process: the acetyltransferase MEC-17, the deacetylase SIRT1, and the acetyl-lysine reader DPF2. Tubulin acetylation by MEC-17 is associated with stable, long-lived microtubule structures. We determined the crystal structure of the catalytic domain of human MEC-17 in complex with the cofactor acetyl-CoA. The structure in combination with an extensive enzymatic analysis of MEC-17 mutants identified residues for cofactor and substrate recognition and activity. A large, evolutionarily conserved hydrophobic surface patch distal to the active site was shown to be necessary for catalysis, suggesting that specificity is achieved by interactions with the alpha-tubulin substrate that extend outside of the modified surface loop. Experiments in C. elegans showed that while MEC-17 is required for touch sensitivity, MEC-17 enzymatic activity is dispensible for this behavior. SIRT1 deacetylates a wide range of substrates, including p53, NF-kappaB, FOXO transcription factors, and PGC-1-alpha, with roles in cellular processes ranging from energy metabolism to cell survival. SIRT1 activity is uniquely controlled by a C-terminal regulatory segment (CTR). Here we present crystal structures of the catalytic domain of human SIRT1 in complex with the CTR in an apo form and in complex with a cofactor and a pseudo-substrate peptide. The catalytic domain adopts the canonical sirtuin fold. The CTR forms a beta-hairpin structure that complements the beta-sheet of the NAD^+-binding domain, covering an essentially invariant, hydrophobic surface. A comparison of the apo and cofactor bound structures revealed conformational changes throughout catalysis, including a rotation of a smaller subdomain with respect to the larger NAD^+-binding subdomain. A biochemical analysis identified key residues in the active site, an inhibitory role for the CTR, and distinct structural features of the CTR that mediate binding and inhibition of the SIRT1 catalytic domain. DPF2 represses myeloid differentiation in acute myelogenous leukemia. Finally, we solved the crystal structure of the tandem PHD domain of human DPF2. We showed that DPF2 preferentially binds H3 tail peptides acetylated at Lys14, and binds H4 tail peptides with no preference for acetylation state. Through a structural and mutational analysis we identify the molecular basis of histone recognition. We propose a model for the role of DPF2 in AML and identify the DPF2 tandem PHD finger domain as a promising novel target for anti-leukemia therapeutics.

Dynamic response of hysteretic systems with application to a system containing limited slip

A general class of single degree of freedom systems possessing rate-independent hysteresis is defined. The hysteretic behavior in a system belonging to this class is depicted as a sequence of single-valued functions; at any given time, the current function is determined by some set of mathematical rules concerning the entire previous response of the system. Existence and uniqueness of solutions are established and boundedness of solutions is examined.

An asymptotic solution procedure is used to derive an approximation to the response of viscously damped systems with a small hysteretic nonlinearity and trigonometric excitation. Two properties of the hysteresis loops associated with any given system completely determine this approximation to the response: the area enclosed by each loop, and the average of the ascending and descending branches of each loop.

The approximation, supplemented by numerical calculations, is applied to investigate the steady-state response of a system with limited slip. Such features as disconnected response curves and jumps in response exist for a certain range of system parameters for any finite amount of slip.

To further understand the response of this system, solutions of the initial-value problem are examined. The boundedness of solutions is investigated first. Then the relationship between initial conditions and resulting steady-state solution is examined when multiple steady-state solutions exist. Using the approximate analysis and numerical calculations, it is found that significant regions of initial conditions in the initial condition plane lead to the different asymptotically stable steady-state solutions.

The political econmoy of state government debt : an analysis of constitutional limitations (1961-1990)

Over the past decade, scholarly interest concerning the use of limitations to constrain government spending and taxing has noticeably increased. The call for constitutional restrictions can be credited, in part, to Washington's apparent inability to legislate any significant reductions in government expenditures or in the size of the national debt. At the present time, the federal government is far from instituting any constitutional limitations on spending or borrowing; however, the states have incorporated many controls on revenues and expenditures, the oldest being strictures on full faith and credit borrowing. This dissertations examines the efficacy of these restrictions on borrowing across the states (excluding Alaska) for the period dating from 1961 to 1990 and also studies the limitations on taxing and spending synonymous with the Tax Revolt.

We include socio-economic information in our calculations to control for factors other than the institutional variables that affect state borrowing levels. Our results show that certain constitutional restrictions (in particular, the referendum requirement and the dollar debt limit) are more effective than others. The apparent ineffectiveness of other limitations, such as the flexible debt limit, seem related to the bindingness of the limitations in at least half of the cases. Other variables, such as crime rates, number of schoolage children, and state personal income do affect the levels of full faith and credit debt, but not as strongly as the limitations. While some degree of circumvention can be detected (the amount of full faith and credit debt does inversely affect the levels of nonguaranteed debt), it is so small when compared to the effectiveness of the constitutional restrictions that it is almost negligible. The examination of the tax revolt era limitations yielded quite similar conclusions, with the additional fact that constitutional restrictions appear more binding than statutory ones. Our research demonstrates that constitutional limitations on borrowing can be applied effectively to constrain excessive borrowing, but caution must be used. The efficacy of these restrictions decrease dramatically as the number of loopholes increase.