by Daron Adam Fincham. / Bibliography: leaves [84-99] / Thesis (M.Phil.) -- Chinese University of Hong Kong, 1983

DNA double-strand breaks induced by ionizing radiation are often accompanied by ancillary oxidative base damage that may prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, XLF-dependent nonhomologous end joining of blunt-ended DSB substrates having the oxidatively modified nonplanar base thymine glycol (Tg) at the first (Tg1) , second (Tg2), third (Tg3) or fifth (Tg5) positions from one 3’ terminus was examined in human whole-cell extracts. Tg at the third position had little effect on end-joining even when present on both ends of the break. However, Tg as the terminal or penultimate base was a major barrier to end joining (>10-fold reduction in ligated products) and an absolute barrier when present at both ends. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised from Tg1, Tg2 and Tg3 largely if not exclusively after DSB ligation. However, Tg was rapidly excised from the Tg5 substrate, resulting in a reduced level of DSB ligation, as well as slow concomitant resection of the opposite strand. XLFL115D mutant completely eliminates ligation of all five substrates and previous X‑ray crystallography shows that XLF binds to XRCC4 via a “leucine lock” motif wherein L115 of XLF slips into a hydrophobic pocket in XRCC4. This makes the XRCC4-XLF interaction a good target to develop peptide inhibitors in order to radiosensitize breast tumor cells that are dependent on NHEJ to repair their DSBs after ionizing radiation exposure. Using mRNA display, we created a diverse library of 870 billion unique peptide sequences. After seven rounds of in vitro selection, the eluted fusions were cloned and sequenced. The results showed homology of sequences of five main families. We have selected representative peptides from those families (Pep 7.1-7.5), and several were chemically synthesized. However, none of these significantly inhibited XLF-dependent end joining in whole-cell extracts. Overall, the results suggest that promoting ligation of DSBs with proximal base damage may be an important function of XLF, but that Tg can still be a major impediment to repair, being relatively resistant to both trimming and ligation. The effectiveness of XLF-XLRCC4 inhibitors in blocking nonhomologous end joining remains to be determined.

<p>α₁-proteinase inhibitor (α₁-PI) is the most abundant serine protease inhibitor (serpin) in plasma. The α₁-PI M358R mutant exhibits greatly increased rates of thrombin inhibition compared to wild type α₁-PI, which predominantly inhibits neutrophil elastase. M358R (P1) lies at the reactive centre (P1-P1’) bond of the reactive centre loop (RCL) of α₁-PI, cleaved by cognate proteases as they become trapped in the serpin-type inhibitory complex. The relationship between RCL structure and serpin inhibitor function is incompletely understood and has not been subjected to saturation mutagenesis. α₁-PI M358R is a less potent inhibitor of thrombin than natural thrombin-inhibitory serpins, suggesting room for engineered improvement into an antithrombotic protein drug.</p> <p>Phage display is a powerful tool for screening mutant protein libraries, but only one serpin (PAI-1) has previously been mutated and expressed in this manner. In this study the T7Select10-3b (Novagen) phage display system was used to express α₁-PI variants and PAI-1, fused to the first 348 residues of the T7 10B coat protein. Following confirmation that α₁-PI M358R retained inhibitory activity when fused to T7Select10-3b phage, this system was used to express a library of α₁-PI mutant proteins with all possible codon combinations at positions P2 (P357) and P1 (M358) (441 mutants). The library was biopanned using a novel technique in order to amplify only the α₁-PI P2P1 mutants capable of forming stable complexes with thrombin. The P357/M358R mutant was the only P2P1 mutant enriched, indicating that the α₁-PI M358R protein has the optimal P2P1 sequence for thrombin inhibition.</p> <p>A second T7Select10-3b library of α₁-PI mutant proteins was generated to identify the optimal sequence at positions P7 through to P3 (amino acids 352-356) for thrombin inhibition. The P2 and P1 positions were maintained at P357/M358R, while all possible codon combinations at positions P7 through to P3 were represented (>4.08 million mutants). The library was biopanned using the protocol developed for the P2P1 library, before sequences were inserted into an <em>E. coli</em> expression vector and α₁-PI M358R P7-P3 mutants were screened for thrombin inhibitory activity. 80 individual colonies were screened, yielding 22 unique P7-P3 mutants with thrombin inhibitory activity greater than the M358R RCL sequence. The consensus observed in sequences with improved activity matched thrombin’s known substrate specificity and also general RCL trends: P7-Not Aromatic/P6-Hydrophobic/P5-T or S/P4-Hydrophobic/P3-Not Aromatic.</p> <p>Kinetic characterization of selected mutants with improved thrombin inhibitory activity yielded two mutants, P7-P3 sequence DITMA and AAFVS, with a second order rate constant of 1.0 x 10⁶ M^-1s^-1. This represents a >2-fold increase in the rate of thrombin inhibition versus α₁-PI M358R. Both the DITMA and AAFVS mutants were found to have a lower stoichiometry of inhibition compared to α₁-PI M358R, indicating that an improved thrombin inhibitory mechanism was also enriched during biopanning.</p> <p>These findings suggest that based on the scaffold of the α₁-PI protein, improved thrombin inhibitory activity can be engineered and selected via phage display. Additionally, this work represents a proof-of-principle for the application of this system to screen libraries of up to 10 million mutants in order to better engineer serpins towards a desired activity.</p> / Master of Health Sciences (MSc)

The present study was divided between two different drug delivery goals, each involving naltrexone (NTX) or its active metabolite, 6-β-naltrexol (NTXOL). First, amino acid esters of NTX and NTXOL were prepared in order to test their candidacy for microneedle-enhanced transdermal delivery. Second, a 3-O-(-)-cytisine-naltrexone (CYT-NTX) codrug was prepared for screening as a potential oral delivery form of NTX and (-)-cytisine (CYT). The amino acid prodrugs were intended for the treatment of alcohol abuse, while the codrug was designed as a single agent for the treatment of alcoholism and tobacco-dependency co-morbidities. One hypothesis of this work was that prodrugs of NTX or NTXOL can be designed that possess superior skin transport properties through microneedle-treated skin compared to parent NTX or NTXOL. Nine amino acid ester prodrugs were prepared, and only three 6-O amino acid ester prodrugs of NTXOL were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. 6-O-β-Ala-NTXOL, the lead compound, exhibited the most rapid bioconversion to NTXOL in human plasma (t1/2 = 2.2 ± 0.1 h); however, this in vitro stability value indicates that the prodrug may require hepatic enzyme-mediated hydrolysis for sufficiently rapid bioconversion to NTXOL in vivo. A second hypothesis of this work was that a CYT-NTX codrug could be designed with appropriate stability characteristics for oral delivery. CYT-NTX was found to be stable over the time course of 24 h in buffer systems of pH 1.5, 5.0, 7.4 and 9.0, and in 80% rat plasma, 80% human plasma, simulated gastric fluid and simulated intestinal fluid. Six (3 rats/group) Sprague-Dawley male rats were dosed i.v. with 1 mg/kg CYT-NTX codrug, or 10 mg/kg, p.o. Oral administration of a 10 mg/kg dose of CYT-NTX codrug resulted in rapid absorption and distribution (5 min) of CYT-NTX codrug, and NTX was released from codrug with a peak plasma concentration of 6.8 ± 0.9 nmol/L reached within 65 minutes. Plasma CYT was not detected; however, NTX delivery was achieved with a fraction absorbed value of 13%. Thus, CYT-NTX may hold promise as a potential oral codrug for further optimization and development.

While protein α-N-terminal methylation has been known for nearly four decades since it was first uncovered on bacteria ribosomal proteins L33, the function of this modification is still not entirely understood. Recent discoveries have demonstrated α-N-terminal methylation is essential to stabilize the interactions between regulator of chromosome condensation 1 (RCC1) and chromatin during mitosis, to localize and enhance the interaction of centromere proteins (CENPs) with chromatin, and to facilitate the recruitment of DNA damage-binding protein 2 (DDB2) to DNA damage foci. Identification of N-terminal methyltransferase 1 (NTMT1) unveiled the eukaryotic methylation writer for protein α-N-termini. In addition, NTMT2 that shares over 50% sequence similarity, has been identified as another mammalian protein α-N-terminal methylation writer. Knockdown of NTMT1 results in mitotic defects and sensitizes chemotherapeutic agents in breast cancer cell lines, while NTMT1 knockout mice showed premature aging. Additionally, NTMT1 has been shown to be overexpressed in a colorectal and melanoma tumor tissues, and in lung and liver cancer cell lines. Given the vast array of clinical relevance, chemical probes and inhibitors for NTMT1 are vital to elucidate information about the function and downstream process of protein α-N-terminal methylation. Therefore, 47 peptidomimetic compounds have been synthesized that target NTMT1. These peptide-based compounds range from three to six amino acids in length and the top 5 compounds have 3- to 300- fold selectivity for NTMT1 compared to other methyltransferases. An inhibition mechanism study has also been performed to verify the inhibitors are targeting the NTMT1 peptide binding site. Seven compounds have an IC50 of less than 5 µM and our top inhibitor, BM-47, has an IC50 of 0.32 µM ± 0.06 for NTMT1. To further elucidate information about the NTMTs and their downstream effects, we utilized photoaffinity probes to target these enzymes. Our 6 photoaffinity probes exhibited in a dose- and time-dependent manner. Probe labeling has been shown to be driven by recognition and selectively and competitively label the NTMT writers in a complex cellular mixture. Our results also provided the first indication of substrate preferences among NTMT1/2. Methylated photoaffinity probes were also synthesized to identify novel proteins that recognize a methylated N-terminus and shed light on the function of α-N-terminal methylation.

The error free repair of DNA double strand breaks through the homologous recombinational repair pathway is essential for organisms of all types to sustain life. A detailed structural and mechanistic understanding of this pathway has been the target of intense study since the identification of bacterial recA, the gene whose product is responsible for the catalysis of DNA strand exchange, in 1965. The work presented here began with defining residues that are important for the assembly and stability of the RecA filament, and progressed to the identification of residues critical for the transfer of ATP-mediated allosteric information between subunits in the protein's helical filament structure. My work then evolved to investigate similar mechanistic details concerning the role of ATP in the human RecA homolog, Rad51. Results from non-conservative mutagenesis studies of the N-terminal region of one subunit and the corresponding interacting surface on the neighboring subunit within the RecA protein, led to the identification of residues critical for the formation of the inactive RecA filament but not the active nucleoprotein filament. Through the use of specifically engineered cysteine substitutions we observed an ATP-induced change in the efficiency of cross subunit disulfide bond formation and concluded that the position of residues in this region as defined by the current crystal structure may not accurately reflect the active form of the protein. These ATP induced changes in positioning led to the further investigation of the allosteric mechanism resulting in the identification of residue Phe217 as the key mediator for ATP-induced information transfer from one subunit to the next. In transitioning to investigate homologous mechanisms in the human pathway I designed a system whereby we can now analyze mutant human proteins in human cells. This was accomplished through the use of RNA interference, fluorescent transgenes, confocal microscopy and measurements of DNA repair. In the process of establishing the system, I made the first reported observation of the cellular localization of one of the Rad51 paralogs, Xrcc3, before and after DNA damage. In addition we found that a damage induced reorganization of the protein does not require the presence of Rad51 and the localization to DNA breaks occurs within 10 minutes. In efforts to characterize the role of ATP in human Rad51 mediated homologous repair of double strand breaks we analyzed two mutations in Rad51 specifically affecting ATP hydrolysis, K133A and K133R. Data presented here suggests that, in the case of human cells, ATP hydrolysis and therefore binding, by Rad51 is essential for successful repair of induced damage.

Calbindin-D28K is a cytosolic calcium binding protein found in a variety of cells that transport calcium. The chorioallantoic membrane and yolk sac of oviparous squamate reptiles (lizards and snakes) transport calcium from the eggshell and yolk to the developing embryo. I used immunohistochemistry to localize calbindin-D28K expression in the chorioallantoic membrane and yolk sac of two species of oviparous scincid lizards, Plestiodon fasciatus and Saproscincus mustelinus. Calbindin-D28K was detected in the chorioallantoic membrane and yolk sac of both lizard species by a polyclonal anti-snake calbindin antibody and a monoclonal anti-cow calbindin antibody. Calbindin-D28K was localized in the chorionic epithelium and allantoic epithelium of the chorioallantoic membrane and in endodermal cells scattered throughout the yolk mass of both species. This is the first demonstration of calbindin-D28K in allantoic epithelium and in endodermal cells of the yolk sac of lizards.

Angiogenesis, the formation of new vessels from a preexisting vasculature, is critical for supplying a healing myocardium with oxygen and nutrients to sustain metabolism post myocardial infarction (MI). Interleukin-1β (IL-1β), a proinflammatory cytokine increased in the heart post-MI, is considered essential for angiogenesis in tumor growth and metastasis, arthritis, endometriosis, and wound healing. Matrix metalloproteinases (MMPs) are implicated in angiogenesis because of their ability to selectively degrade components of the extracellular matrix. Vascular endothelial growth factors (VEGFs) play a vital role in angiogenesis because of their involvement in the recruitment and proliferation of endothelial cells. The current study explores IL-1β-stimulated regulation of angiogenic genes in cardiac microvascular endothelial cells (CMECs), the signaling mechanisms involved, and the implications in the processes of angiogenesis. DNA microarray analysis indicated IL-1β modulates the expression of numerous angiogenesis-related genes, notably upregulating MMP-2 and downregulating VEGF-D expression. RT-PCR and Western blot analyses confirmed the differential expression in response to IL-1β. In-gel zymographic analysis demonstrated IL-1β-stimulated increase in MMP-2 activity. IL-1β activated ERK1/2 and JNKs, not p38 kinase, and activated PKCα/β1 independent of MAPKs. IL-1β inactivated GSK3β via ERK1/2. Pharmacological inhibition of these signaling cascades indicated IL-1β-stimulated regulation of MMP-2 and VEGF-D occurs via ERK1/2, JNKs, and PKCα/β1-dependent mechanisms. In addition, inactivation of GSK3β inhibited basal VEGF-D expression. H2O2 significantly increased MMP-2 protein levels while IL-1β-induced VEGF-D downregulation was further potentiated by ROS scavenging compounds and inhibition of NF-κB. Phalloidin-FITC stain indicated a sharp reduction in fibrillar actin in the cytoskeleton of IL-1β-stimulated cells. Wounding assays revealed that IL-1β induced CMEC migration but prevented cell-to-cell contact and restoration of the monolayer. Flow cytometric analysis revealed a G0/G1 phase cell cycle arrest in IL-1β-stimulated cells, indicative of decreased proliferation. IL-1β inhibited three-dimensional in vitro tube formation by CMECs. Lastly, IL-1β inhibited microvessel sprouting from aortic rings, an assay examining the collective response of multiple cell types. Collectively, the data presented in this study provide evidence that IL-1β differentially regulates important angiogenesis-related genes in CMECs. This differential regulation may lead to interruptions in the processes of angiogenesis, ultimately creating a dysfunctional phenotype for myocardial vessel formation.

Platinum complexes (cisplatin, carboplatin and oxaliplatin) are effective anticancer agents. However the major drawbacks of platinum chemotherapy are toxic side effects and resistance. The affinity of platinum complexes to sulfur donor ligands of side chains of methionine and cysteine amino acids was assumed to be responsible for toxicity and resistance. Recently, it was found that the reaction of platinum complex with proteins containing sulfur donor ligands could actually favor its anticancer activity. Copper transporter 1 (Ctr 1), a protein involved in the transport of copper into the cell, also helps in the influx of cisplatin by binding to N-terminal domain of Ctr 1 which is rich in methionine and histidine residues. A better understanding of how the size and shape of amine ligand, and leaving groups affect the reaction of platinum (II) complexes with methionine could give new ways to optimize its anticancer activity. This preliminary research focuses to answer this by HPLC-UV-VIS analysis of bulky platinum complexes including [Pt(dien)Cl]Cl, Pt(Me4en)(NO3)2 and Pt(en)(NO3)2 with two methionine containing small peptides that serve as models for protein interactions.

Cytoplasmic dynein is a multisubunit complex involved in retrograde transport of cellular components along microtubules. The heavy chains (HC) are very large catalytic subunits which possess microtubule binding ability. The intermediate chains (IC) are responsible for targeting dynein to its appropriate cargo by interacting with the dynactin complex. The light intermediate chains (LIC) are previously unexplored subunits that have been proposed to modulate dynein activity by regulating the motor or the IC-dynactin interaction. The light chains (LC) are a newly identified class of subunit which are also thought to have regulatory functions. In the first part of this work, I analyzed the relationship between the four SDS-PAGE gel bands that comprise the light intermediate chains. 1- and 2-D electrophoresis before and after alkaline phosphatase treatment revealed that the four bands are derived from two different polypeptides, each of which is phosphorylated. Peptide microsequencing of these subunits yielded sequences that indicated similarity between them. cDNA cloning of the rat LICs revealed the presence of a conserved P-loop sequence and a very high degree of homology between the two different rat LICs and among LICs from different species. The second series of experiments was designed to analyze the association of pericentrin with cytoplasmic dynein. First, various dynein and dynactin subunits were co-associate with pericentrin in these experiments. Co-precipitation from 35S labeled cell extracts revealed a direct interaction between LIC and pericentrin. Comparison of pericentrin binding by LICl and LIC2 showed that only LICl was able to bind. Further investigation of the relationship between LICl and LIC2 demonstrated that each LIC will self-associate, but they will not form heterooligomers. Additionally, using co-overexpression and immunoprecipitation of LICl, LIC2, and HC, I have shown that binding of the two LICs to HC is mutually exclusive. Finally, I investigated the relationships between dynein HC, IC, and LIC by examining the interactions among the subunits. IC and LIC were both found to bind to the HC, but not to each other. Despite the lack of interaction between IC and LIC, they are, in fact, present in the same dynein complexes and they have partially overlapping binding sites within the N-terminal sequence of the HC. The HC dimerization site was determined to extend through a large portion of the N-terminus, and it includes both the IC and LIC binding sites, although these subunits are not required for dimerization. Together these studies implicate the light intermediate chains in dynein targeting. Targeting of dynein to its cargo has been thought to be performed by the dynactin complex, and for one particular cargo, the kinetochore, there is considerable evidence to support this model. The results presented here suggest that the light intermediate chains appear to function in a separate, non-dynactin-based targeting mechanism.