Poly(ADP)-Ribose Polymerase Activity in the Eukaryotic Mono-ADP-Ribosyl Transferase, ART2: a Dissertation

The glycophosphatidylinositol(GPI)-linked membrane protein ART2 is an antigenic determinant for T lymphocytes that regulate the expression of diabetes in the BB/W rat model. Though little is understood of the physiologic role of ART2 on T lymphocytes, ART2 is a member of the mono-ADP-ribosyl transferase subgroup ofthe ADP-ribosyl transferase (ART) protein family. The ART protein family, which traditionally has been divided into mono-ADP-ribosyl transferases (mono-ARTs), poly(ADP)-ribose polymerases (PARPs), and ADP-ribosyl cyclases, influences various aspects of cellular physiology including: apoptosis, DNA damage repair, chromatin remodeling, telomere replication, cellular transport, immune regulation, neuronal function, and bacterial virulence. A structural alignment of ART2.2 with chicken PARP indicated the potential for ART2.2 to catalyze ADP-ribose polymers in an activity thought to be specific to the PARP subgroup and important for their regulation of nuclear processes. Kinetic studies determined that the auto-ADP-ribosyl transferase activity of ART2.2 is multitmeric and heterogeneous in nature. Hydroxylamine-cleaved ADP-ribose moieties from the ART2.2 multimers ran as polymers on a modified sequencing gel, and digestion of the polymers with snake-venom phosphodiesterase produced AMP and the poly(ADP)ribose-specific product, PR-AMP, which was resolved by analytical HPLC and structurally confirmed by ESI-MS. The ratio of AMP to PR-AMP was higher than that of PARP raising the possibility that the ART2.2 polymers had a different branching structure than those of PARP. This alternative branching was confirmed by the presence of ribose phosphate polymers in the snake venom phophodiesterase treated samples. The site of the auto-poly(ADP)-ribose modification was determined to be R185, a residue previously proposed to influence the level of auto-ADP ribosylation of ART2.2 by mutational analysis. These data provide the first demonstration of a hybrid between mono-ARTs and PARPs and are the earliest indication that PARP-like enzymes can exist outside the nucleus and on the cell surface

The Regulation of nNOS During Neuronal Differentiation and the Effect of Nitric Oxide on Hdm2-p53 Binding: a Dissertation

Nitric oxide is a ubiquitous signaling molecule with both physiological and pathological functions in biological systems. Formed by the enzymatic conversion of arginine to citrulline, NO, has known roles in circulatory, immune and nervous tissues. In the nervous system nitric oxide has been implicated in long-term potentiation, neurotransmitter release, channel function, neuronal protection and neuronal degeneration. Much of our work has focused on yet another role for nitric oxide in cells, namely, neuronal differentiation. During development, neuronal differentiation is closely coupled with cessation of proliferation. We use nerve growth factor (NGF)-induced differentiation of PC12 pheochromocytoma cells as a model and find a novel signal transduction pathway that blocks cell proliferation. Treatment of PC12 cells with NGF leads to induction of nitric oxide synthase (NOS). The resulting nitric oxide (NO) acts as a second messenger, activating the p21(WAF1) promoter and inducing expression of p21(WAF1) cyclin-dependent kinase inhibitor. NO activates the p21(WAF1) promoter by p53-dependent and p53-independent mechanisms. Blocking production of NO with an inhibitor of NOS reduces accumulation of p53, activation of the p21(WAF1) promoter, expression of neuronal markers, and neurite extension. To deternine whether p21(WAF1) is required for neurite extension, we prepared a PC12 line with an inducible p21(WAF1) expression vector. Blocking NOS with an inhibitor decreases neurite extension, but induction of p21(WAF1) with isopropyl-1-thio-beta-D-galactopyranoside restored this response. Levels of p21(WAF1) induced by isopropyl-1-thio-beta-D-galactopyranoside were similar to those induced by NGF. Therefore, we have identified a signal transduction pathway that is activated by NGF; proceeds through NOS, p53 and p21(WAF1) to block cell proliferation; and is required for neuronal differentiation by PC12 cells. In further studies of this pathway, we have examined the role of MAP kinase pathways in neuronal nitric oxide synthase (nNOS) induction during the differentiation of PC12 cells. In NGF-treated PC12 cells, we find that nNOS is induced at RNA and protein levels, resulting in increased NOS activity. We note that neither nNOS mRNA, nNOS protein nor NOS activity is induced by NGF treatment in cells that have been infected with a dominant negative Ras adenovirus. We have also used drugs that block MAP kinase pathways and assessed their ability to inhibit nNOS induction. Even though U0126 and PD98059 are both MEK inhibitors, we find that U0126, but not PD98059, blocks nNOS induction and NOS activity in NGF-treated PC12 cells. Also, the p38 kinase inhibitor, SB 203580, does not block nNOS induction in our clone of PC12 cells. Since the JNK pathway is not activated in NGF-treated PC12 cells, we determine that the Ras-ERK pathway and not the p38 or JNK pathway is required for nNOS induction in NGF-treated PC12 cells. We find that U0l26 is much more effective than PD98059 in blocking the Ras-ERK pathway, thereby explaining the discrepancy in nNOS inhibition. We conclude that the Ras-ERK pathway is required for nNOS induction. The activation of soluble guanylate cyclase and the production of cyclic GMP is one of the best characterized modes of NO action. Having shown that inhibition of NOS blocks PC12 cell differentiation we tested whether nitric oxide acts through soluble guanylate cyclase to lead to cell cycle arrest and neuronal differentiation. Unlike NOS inhibition, the inhibition of soluble guanylate cylcase does not block the induction of neuronal markers. Moreover, treatment of NGF-treated, NOS-inhibited PC12 cells with a soluble analog of cyclic GMP was unable to restore differentiation of those cells. Hence, cGMP is not a component of this pathway and we had to consider other mechanisms of NO action. It has become increasingly evident that another manner by which NO may exert its effects is by S-nitrosylation of cysteine residues. We tested, in vitro whether nitric oxide may control p53 by S-nitrosylation and inactivation of the p53 negative regulator, Hdm2. Treatment of Hdm2 with a nitric oxide donor inhibits Hdm2-p53 binding, the first step in Hdm2 regulation of p53. The presence of cysteine or DTT blocks this inhibition of binding. Moreover, nitric oxide inhibition of Hdm2-p53 binding was found to be reversible. Sulfhydryl-sensitivity and reversibility are consistent with nitrosylation. Finally, we have identified a critical cysteine residue that nitric oxide modifies in order to disrupt Hdm2-p53 binding. Mutation of this residue from a cysteine to an alanine does not interfere with binding but rather eliminates the sensitivity of Hdm2 to nitric oxide inactivation

Probing the dNTP Binding Region of \u3cem\u3eBacillus subtilis\u3c/em\u3e: DNA Polymerase III with Site-Directed Inhibitors: A Dissertation

6-(p-Hydroxyphenylhydrazino) uracil (H2-HPUra) is a selective and potent inhibitor of the replication-specific DNA polymerase III (pol III) of Gram+ bacteria such as Bacillus subtilis. Although a pyrimidine, H2-HPUra derives its inhibitory activity from its specific capacity to mimic the purine nucleotide, dGTP. The project described in this thesis dissertation involves the use of H2-HPUra-like inhibitors to probe the structure and function of the pol III active site. It consists of two separate problems which are summarized below. Production of a potent bona fide dGTP form of inhibitor. A method was devised to successfully convert the H2-HPUra inhibitor prototype to a bona fide purine, using N2-benzyl guanine as the basis. Structure-activity relationships of benzyl guanines carrying a variety of substituents on the aryl ring identified N2-(3,4-dichlorobenzyl) guanine (DCBG) as a compound equivalent to H2-HPUra with respect to potency and inhibitor mechanism. DCBdGTP, the 2\u27-deoxyribonucleoside 5\u27-triphosphate form of DCBG, was synthesized and characterized with respect to its action on wild-type and mutant forms of pol III. DCBdGTP acted on pol III by the characteristic inhibitor mechanism and formally occupied the dNTP binding site with a fit which permitted its polymerization. The latter experiment identified the site for the binding of the inhibitor\u27s aryl moiety as a distinct site located at a distance of approximately 6-7 Å from the base-paired 2-NH group of a bound dGTP. Attempt to covalently label amino acid residue 1175, a putative participant in inhibitor binding. Azp-12, a point mutation of serine 1175, yields a form of pol III whose inhibitior sensitivity varies specifically as a function of the composition of the para substituent of the inhibitor\u27s aryl ring. On the basis of the latter behavior, residue 1175 was hypothesized to be a residue directly involved in the binding of the inhibitor\u27s aryl moiety. To test this hypothesis, residue 1175 was specifically mutated to either cysteine or lysine, each of which presents a side chain amenable to covalent bond formation with appropriately reactive inhibitor forms. Of the two mutant pol III forms, only the cysteine form (pol III-cys) was catalytically active. The kinetic properties and inhibitor sensitivity profile of pol III-cys identified it as a target suitable for potentially irreversible inhibitor forms containing the following groups in the meta position of the aryl ring: -CH2Br, -CH2C1, and -CH2SH. None of the several inhibitors tested selectively or irreversibly inactivated pol III-cys. Possible bases for the failure of this group of inhibitors and for the redesign of more useful covalently reactive inhibitor forms are considered

The Structure, Function, and Regulation of Insulin-like Growth factor II/Mannose 6-phosphate Receptor Forms: a Thesis

In mammals a single receptor protein binds both insulin-like growth factor II (IGF-II) and mannose 6-phosphate (Man 6-P) containing ligands, most notably lysosomal enzymes. However, in chick embryo fibroblasts IGF-II binds predominantly to a type 1 IGF receptor, and no IGF-II/Man 6-P receptor has been identified in this species. In order to determine if chickens possess an IGF-II/Man 6-P receptor, an affinity resin (pentamannosyl 6-phosphate (PMP) Sepharose) was used to purify receptors from chicken membrane extracts by their ability to bind mannose 6-phosphate. Then 125I-IGF-II was used to evaluate their ability to bind IGF-II. These experiments demonstrate that nonmammalian Man 6-P receptors lack the ability to bind IGF-II, suggesting that the ability to bind IGF-II has been gained recently in evolution by the mammalian Man 6-P receptor. The second area of study involves the serum form of the IGF-II/Man 6-P receptor. This receptor had been detected in the serum of a number of mammalian species, yet its structure, function, regulation, and origin were unknown. Initial studies, done with Dr. R. G. MacDonald, showed that the serum receptor is truncated such that the C-terminal cytoplasmic domain of the cellular receptor is removed. These studies also demonstrate a regulation of serum receptor levels with age, similar to that seen for the cellular receptor, and that the serum form of the receptor existed in several forms which appeared intact under nonreducing conditions, but as multiple proteolytic products upon reduction. Finally, these studies demonstrated that both the cellular and serum IGF-II/Man 6-P receptors are capable of binding IGF-II and Man 6-P simultaneously. In studies on the serum form of the IGF-II/Man 6-P receptor that I have conducted independently, the regulation of the serum IGF-II/Man 6-P and transferrin receptors by insulin has been demonstrated. In these studies, insulin injected into rats subcutaneously resulted in a time and dose dependent increase in serum receptor levels. Finally, to investigate the relationship of the serum IGF- II/Man 6-P receptor to the cellular form of the receptor, pulse chase experiments were performed. These experiments demonstrate that the soluble (serum form released into the medium) receptor is a major degradation product of the cellular receptor. Furthermore, the lack of detectable amounts of the lower Mr soluble receptor intracellularly and the parallel relationship of cell surface and soluble receptor suggest that the proteolysis is occurring from the cell surface. Finally, a number of experiments suggest that the degradation rate depends upon the conformation state of the receptor: binding of IGF-II or Man 6-P makes the receptor more susceptible to proteolysis while the presence of lysosomal enzymes prevents receptor proteolysis. In summary, the serum form of the IGF-II receptor is a proteolytic product of the cellular form of the receptor. The rate of release depends upon the number of receptors at the cell surface and the binding state of the receptor. In circulation, the receptor retains the ability to bind both types of ligands, it thus may serve as an IGF binding protein and/or a lysosomal enzyme binding protein. These results suggest a model whereby the cellular receptor is proteolytically cleaved by a plasma membrane protease to produce a short membrane anchored fragment and the serum receptor. In vivo this pathway serves as the major degradative pathway of the IGF-II/Man 6-P receptor, with the serum form being cleared from circulation by further degradation and reuptake

Association of Pericentrin with the γ Tubulin Ring Complex: a Dissertation

Pericentrin is a molecular scaffold protein. It anchors protein kinases, (PKB, (Purohit, personal communication), PKC, (Chen et al., 2004), PKA Diviani et al., 2000), the γ tubulin ring complex, (γ TuRC) (Zimmerman et al., 2004), and possibly dynein (Purohit et al., 1999) to the spindle pole. The γ TuRC is a ~ 2 MDa complex which binds the minus ends of microtubules and nucleates microtubules in vitro, (Zheng et al., 1995). Prior to this work, nothing was known about the association of the γTuRC with pericentrin. Herein I report the biochemical identification of a large protein complex in Xenopus extracts containing pericentrin, the γ TuRC, and other as yet unidentified proteins. Immunodepletion of γ tubulin results in co-depletion of pericentrin, indicating that virtually all the pericentrin in a Xenopus extract is associated with γ tubulin. However, pericentrin is not a member of the, γ TuRC, since isolated γ TuRCs do not contain pericentrin. The association of pericentrin with the γ TuRC is readily disrupted, resulting in two separable complexes, a small pericentrin containing complex of approximately 740 KDa and the the γ TuRC, 1.9 MDa in Xenopus. Co overexpression/ coimmunoprecipitation and yeast two hybrid studies demonstrate that pericentrin binds the γTuRC through interactions with both GCP2 and GCP3. When added to Xenopus mitotic extracts, the GCP2/3 binding domain uncoupled γ TuRCs from centrosomes, inhibited microtubule aster assembly and induced rapid disassembly of pre-assembled asters. All phenotypes were significantly reduced in a pericentrin mutant with diminished GCP2/3 binding, and were specific for mitotic centro somal asters as I observed little effect on interphase asters or on asters assembled by the Ran-mediated centrosome-independent pathway. Overexpression of the GCP2/3 binding domain of pericentrin in somatic cells perturbed mitotic astral microtubules and spindle bipolarity. Likewise pericentrin silencing by small interfering RNAs in somatic cells disrupted γ tubulin localization and spindle organization in mitosis but had no effect on γ tubulin localization or microtubule organization in interphase cells. Pericentrin silencing or overexpression induced G2/antephase arrest followed by apoptosis in many but not all cell types. I conclude that pericentrin anchoring of γ tubulin complexes at centrosomes in mitotic cells is required for proper spindle organization and that loss of this anchoring mechanism elicits a checkpoint response that prevents mitotic entry and triggers apoptotic cell death. Additionally, I provide functional and in vitro evidence to suggest that the larger pericentrin isoform (pericentrin B/ Kendrin) is not functionally homologous to pericentrin/pericentrin A in regard to it\u27s interaction with the γ TuRC

Mechanistic Analysis of Chromatin Remodeling Enzymes: a Dissertation

The inherently repressive nature of chromatin presents a sizeable barrier for all nuclear processes in which access to DNA is required. Therefore, eukaryotic organisms ranging from yeast to humans rely on a battery of enzymes that disrupt the chromatin structure as a means of regulating DNA transactions. These enzymes can be divided into two broad classes: those that covalently modify histone proteins, and those that actively disrupt nucleosomal structure using the free energy derived from ATP hydrolysis. The latter group, huge, multisubunit ATP-dependent chromatin remodeling factors, are emerging as a common theme in all nuclear processes in which access to DNA is essential. Although transcription is the process for which a requirement for chromatin remodeling is best documented, it is now becoming clear that other processes like replication, recombination and DNA repair rely on it as well. A growing number of ATP-dependent remodeling machines has been uncovered in the last 10 years. Although they differ in their subunit composition, organism or tissue restriction, substrate specificity, and regulating/recruiting partners, it has become increasingly evident that all ATP-dependent chromatin remodeling factors share a similar underlying mechanism. This mechanism is the subject of the studies presented in this thesis. Chromatin-remodeling factors seem to bind both the histone and DNA components of nucleosomes. From a fixed position on nucleosomes, the remodeling factors appear to translocate on the DNA, generating torsional stress on the double helix. This activity has several consequences, including the distortion of the DNA structure on the surface of the histone octamer, the disruption of histone-DNA interactions, and the mobilization of the nucleosome core with respect to the DNA. The work presented in this thesis, along with data reported by other groups, supports the hypothesis that yeast SWI/SNF chromatin remodeling complex and the recombinational repair factor, Rad54p, both employ similar mechanisms to regulate gene transcription, and facilitate homologous DNA pairing and recombination, respectively

Soil-Structure Modeling and Design Considerations for Offshore Wind Turbine Monopile Foundations

Offshore wind turbine (OWT) support structures account for 20-25% of the capital cost for offshore wind installations, making it essential to optimize the design of the tower, substructure, and foundation to the extent possible. This dissertation focuses on monopile foundations, as the vast majority (approximately 75%) of currently installed OWTs are supported by monopile structures. The objective of this dissertation is to provide information on the behavior of monopile support structures to better substantiate design and planning decisions and to provide a basis for reducing the structural material costs. In pursuit of these objectives, research is presented on the topics of hysteretic soil-structure damping (referred to as foundation damping), cyclic degradation of soil properties, and the impact of marine growth on OWT monopile support structures. OWTs are lightly damped structures that must withstand highly uncertain offshore wind and wave loads. In addition to stochastic load amplitudes, the dynamic behavior of OWTs must be designed with consideration of stochastic load frequency from waves and mechanical load frequencies associated with the spinning rotor during power production. The close proximity of the OWT natural frequency to excitation frequencies combined with light damping necessitates a thorough analysis of various sources of damping within the OWT system; of these sources of damping, least is known about the contributions of damping from soil-structure interaction (foundation damping), though researchers have back-calculated foundation damping from “rotor-stop” tests after estimating aerodynamic, hydrodynamic, and structural damping with numerical models. Because design guidelines do not currently recommend methods for determining foundation damping, it is typically neglected. The significance of foundation damping on monopile-supported OWTs subjected to extreme storm loading was investigated using a linear elastic two-dimensional finite element model. A simplified foundation model based on the soil-pile mudline stiffness matrix was used to represent the monopile, and hysteretic energy loss in the foundation was converted into a viscous, rotational dashpot at the mudline to represent foundation damping. The percent critical damping contributed to the OWT structural system by foundation damping was quantified using the logarithmic decrement method on a finite element free vibration time history, and stochastic time history analysis of extreme storm conditions indicated that mudline OWT foundation damping can significantly decrease the maximum and standard deviation of mudline moment. Further investigation of foundation damping on cyclic load demand for monopile-supported OWTs was performed considering the design situations of power production, emergency shutdown, and parked conditions. The NREL 5MW Reference Turbine was modeled using the aero-hydro-elastic software FAST and included linear mudline stiffness and damping matrices to take into account soil-structure interaction. Foundation damping was modeled using viscous rotational mudline dashpots which were calculated as a function of hysteretic energy loss, cyclic mudline rotation amplitude, and OWT natural frequency. Lateral monopile capacity can be significantly affected by cyclic loading, causing failure at cyclic load amplitudes lower than the failure load under monotonic loading. For monopiles in clay, undrained clay behavior under short-term cyclic soil-pile loading (e.g. extreme storm conditions) typically includes plastic soil deformation resulting from reductions in soil modulus and undrained shear strength which occur as a function of pore pressure build-up. These impacts affect the assessment of the ultimate and serviceability limit states of OWTs via natural frequency degradation and accumulated permanent rotation at the mudline, respectively. Novel combinations of existing p-y curve design methods were used to compare the impact of short-term cyclic loading on monopiles in soft, medium, and stiff clay. Marine growth increases mass and surface roughness for offshore structures, which can reduce natural frequency and increase hydrodynamic loads, and can also interfere with corrosion protection and fatigue inspections. Design standards and guidelines do not have a unified long-term approach for marine growth on OWTs, though taking into account added mass and increased drag is recommended. Some standards recommend inspection and cleaning of marine growth, but this would negate the artificial reef benefits which have been touted as a potential boon to the local marine habitat. The effects of marine growth on monopile-supported OWTs in terms of natural frequency and hydrodynamic loading are examined, and preliminary recommendations are given from the engineering perspective on the role of marine growth in OWT support structure design