Adrenocorticotropic hormone (ACTH) is synthesized in the pituitary gland as part of a larger precursor molecule. This precursor is characterized by the fact that it contains within its structure multiple chemical messengers with quite different biological activities. Among them are included the melanotropins (MSH), the beta-lipotropin ((beta)-LPH) and the endorphins. To account for the major biological activities of its components, the name pro-opiomelanocortin (POMC) has been proposed for this common precursor. Characterization of this molecule in pituitary tissues including both the anterior and intermediate lobes as well as the mouse anterior pituitary tumour cell line AtT-20/D16v, established its apparent molecular weight (Mr) to be around 30 000 Daltons with the presence of carbohydrate side chains attached to a peptide backbone. This latter aspect is considered partly responsible for the heterogeneity observed in both the molecular weight and the charge of the molecule. Nucleotide sequencing of the cDNA coding for POMC has predicted the whole primary structure. ACTH and (beta)-LPH together account for only half the size of the precursor, the other half being located on the amino-terminal side. This cryptic segment as ACTH and (beta)-LPH contains an MSH-like sequence called (gamma)-MSH. Pulse and pulse-chase experiments indicated that this cryptic segment of the POMC has an apparent molecular weight of 16 000 to 19 000 Daltons and that it is an important maturation product. / In order to completely characterize this cryptic segment we dissected porcine pituitary glands in both the anterior and neurointermediate lobes. Our objective was to determine the molecular form of the amino terminal segment of the porcine POMC (pNT) in the anterior lobe using a chemical approach. We extracted the peptides from the anterior lobes and chromatographed them on various systems including molecular sieving, ion-exchange and high performance liquid chromatography. Complete primary structure of the pNT revealed that the major form is a glycopeptide of 80 amino acid residues with at least two glycosylation sites. Two other peptides of 107 and 61 amino acid residues were isolated. We proposed that they are members of the genealogic tree of the pNT, the 107 residue being the whole cryptic segment preceding ACTH and the amidated 61 residue being a maturation product of the major secretory form. We also isolated a peptide which links the 80 residue glycopeptide to the ACTH in the POMC; it will be called the joining peptide (pJP).

Age-related differences in the in vivo incorporation of 32-P into mouse myelin basic proteins (MBPs) of the central nervous system were observed. The resulting specific radioactivity (S.A.) of the MBPs appeared to be related to the S.A. of the acid-soluble pool of phosphates of myelin. In development, MBPs were phosphorylated in vivo prior to the onset of myelination in the brain, indicating that MBPs are phosphorylated prior to their deposition in the myelin sheath. / The incorporation of 32-P into MBPs and the turnover rates of MBP phosphates were studied in vivo in developmentally-related myelin compartments. The results suggest that there are two separate events in MBP phosphorylation and that the turnover rates of the MBP phosphates derived from these two events are different. / A model for MBP phosphorylation, that could explain these observations, is postulated and discussed in the light of existing information.

The activities of Neurospora endo-exonuclease, an enzyme which had been implicated in DNA recombination and repair, on double stranded DNAs are characterized further in this work. Endo-exonuclease is shown to have a very low bona fide endonuclease activity on covalently closed circular double stranded DNA. Much higher activity is seen with linear double stranded DNA. The enzyme is shown to bind the DNA at a terminus and track in the 5$ sp prime to 3 sp prime$ direction to make site-specific nicks and double strand breaks at or very near the hexanucleotide sequence pAGCACT or related sequences. The nuclease has a higher affinity for binding DNA with protruding 5$ sp prime$-p termini than for DNA with flush or recessed 5$ sp prime$-p termini. All of the above activities with linear DNA are dependent on the presence of the 5$ sp prime$ terminal phosphates. / A very specific, cytosolic inhibitor of endo-exonuclease has been purified. The inhibitor is a single polypeptide of 24 kDa which is trypsin-sensitive and very heat-stable. The protein inhibits the double strand DNase activity of endo-exonuclease competitively and fully when present in excess. The inhibition of the single strand DNase activity is noncompetitive but incomplete even when the inhibitor is present in excess. The inhibitor is also shown to inhibit the site-specific nicking and breaking activities of endo-exonuclease. The 24 kDa inhibitor is present in the cell in three forms: two free forms and one in association with inactive but trypsin-activatable endo-exonuclease. One of the free forms is shown to be related to an inhibitor which is induced by heat shock. / Several inhibitory polypeptides (with molecular sizes of 94, 83, 52, 35 and 17 kDa) copurify with the 24 kDa inhibitor and are immunochemically related to it. The results of immunochemical studies of these polypeptides indicate that the 24 kDa inhibitor and endo-exonuclease are derived from a common higher molecular weight precursor, probably 94 kDa in size. A role for the inhibitor in the regulation of endo-exonuclease activities is suggested. Finally, a model for the action of endo-exonuclease in recombination and double strand break repair is proposed.

No description available.

The three dimensional structure of P-gp nucleotide binding sites is unknown but a high resolution crystal structure was solved for the homodimer of HiSP, the ATP binding subunit of the bacterial ABC transporter histidine permease. The main goal was to test the hypothesis that the nucleotide binding domains (NBD) of P-gp are structurally and functionally conserved with HisP with respect to key residues in intimate contact with the ATP molecule and possibly involved in catalysis. The strategy adopted involves the insertion of cysteine residues at the desired position in a cysteine-less protein backbone. A set of 14 mutants was generated this way for each NBD. The effects of the mutations on drug transport are tested with a cytotoxicity assay using the anti-fungal drug FK506. A431C/A1074C and T435C/T1078C showed the same level of activity as the wild-type while the other residues were completely or partially defective. (Abstract shortened by UMI.)

Aminoglycosides comprise a class of small molecules that have found their way to being one of the most used antibiotics for fighting bacterial infections. Their effectiveness has, however, been compromised in recent years due to the rise in the number of aminoglycoside-resistant strains of bacteria. Their resistance is conferred by enzymes that modify aminoglycosides that are now ubiquitous in both Gram-positive and Gram-negative bacteria. This has prompted a renewed look towards seeking novel drugs via structure-activity relationships. Aminoglycoside phosphotransferases (APH) phosphorylate hydroxyl moieties present on aminoglycosides that render them ineffective. The majority of APR's exhibits broad substrate specificity. Interestingly, APH(9)-Ia, isolated from Legionella pneumophila, phosphorylates one compound, spectinomycin. To further establish the basis for designing a universal drug for APR's and the rationale behind its substrate specificity, the crystal structure of APH(9)-Ia in its ternary form (with bound ADP and spectinomycin) was solved to a resolution of 2.5 A. The overall architecture reveals a helix (alphaC), characteristically larger than its other APH counterparts, which contains three key residues (Arg284, Asp288, and Tyr292) involved in spectinomycin binding. In addition, there are only two acidic residues that participate in its binding, setting APH(9)-Ia apart from multiaminoglycoside detoxifying enzymes. We propose that these features are responsible for the enzyme's single-substrate profile. Furthermore, the structure reveals a significant lobe movement for substrate binding, previously unseen in this class of enzymes. An inhibition study using eukaryotic protein kinase inhibitor, CKI-7, showed it indeed does inhibit the enzyme (KI = (159 +/- 11) muM), though with 2.5 times less effectiveness than for APH(3')-IIIa. Search for possible alternative substrate for this enzyme showed that APH(9)-Ia does not phosphorylate

Folate-dependent enzymes are compartmentalized between the cytoplasm and mitochondria of eukaryotic cells. The cytoplasm contains an NADP-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolate synthetase that is involved in interconverting one-carbon substituted tetrahydrofolates that play essential roles in thymidylate and purine biosynthesis as well as in methionine regeneration. The extent of contribution of mitochondrial folate metabolism to cytoplasmic processes is an area of active investigation. A bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) is located in mitochondria of certain mammalian cells. NMDMC expression in embryonic tissues and established cell lines but not in most adult tissues suggests that it plays a role in embryonic development. Examination of the expression pattern revealed that nmdmc is widely expressed in tissues of embryonic day (E)9.5 and E10.5 embryos. To determine its metabolic role, a knockout approach was used to disrupt the gene encoding NMDMC in mice. Heterozygous mice appear normal but nmdmc-/- embryos die in utero beyond E13.5. Nmdmc-/- fetal livers at E12.5 are smaller in size, lack the characteristic red coloring of a normal liver, but contain similar frequencies of hematopoietic progenitors as do the livers of wild type or heterozygous littermates. This indicates that the failure to establish definitive erythropoiesis in nmdmc-/- embryos is due to the inability of the hematopoietic progenitors to proliferate. Established fibroblasts were generated from wild type, heterozygous and null mutant embryos. Mitochondria of nmdmc-/- cells are structurally intact, retain a membrane potential and do not require NMDMC to support the initiation of protein synthesis. Null mutant fibroblasts are glycine auxotrophs which demonstrates that NMDMC is the only dehydrogenase/cyclohydrolase (synth

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed enzyme that is involved in multiple signaling pathways. Biochemical and substrate trapping studies have implicated PTP1B in the dephosphorylation of various tyrosine kinases, including the EGFR, PDGFR, IR, IGF-IR, JAK2, p210Bcr-Abl, and Src. Of particular interest, gene-targeting studies in mice have established PTP1B as a critical physiological regulator of metabolism by attenuating insulin and leptin signaling. Indeed, PTP1B null mice exhibit resistance to diet-induced diabetes and obesity. Although PTP1B is involved in signaling pathways that contribute to oncogenesis, PTP1B null mice do not develop spontaneous tumors. Therefore, my doctoral research focuses on identifying the physiological significance of PTP1B in these pathways. Our laboratory has previously demonstrated that PTP1B modulates leptin signaling via the tyrosine kinase JAK2. Accordingly, I have shown that PTP1B dephosphorylates JAK2 in a growth hormone (GH)-dependent manner, thus negatively regulating GH signaling and downstream effectors such as STAT3 and STAT5. Consequently, mice lacking PTP1B remain sensitive to GH action after starvation. In addition, I showed that the absence of PTP1B could improve glycemia during streptozotocin-induced type 1 diabetes. In the second part of my research, I have elucidated the mechanism for the previously reported decreased ERK activation in PTP1B null fibroblasts. I demonstrated that Ras activity is reduced in these cells, which is due to increased p120RasGAP expression and p62Dok hyperphosphorylation. Both of these molecules negatively regulate Ras activity by promoting the intrinsic GTPase activity of Ras, leading to decreased ERK activation. Finally, I developed a mouse model of cancer to study the role of PTP1B in tumorigenesis. Since the majority of cancers harbor mutations in p53, I generated p53/PTP1B double null mice. In the absence of p53, PTP1B heterozygous

The E4orf4 protein of human adenovirus kills transformed cancer cell lines by a p53-independent mechanism. Accumulating evidence suggests that the mechanism of E4orf4-induced death is distinct from apoptosis and occurs via some novel pathway. Previous studies have shown that E4orf4 can interact with the mammalian Balpha subunit of protein phosphatase 2A (PP2A) and that this interaction is important for E4orf4-induced cell death. As PP2A is highly conserved across eukaryotic species we chose to study the effects of E4orf4 in a genetically tractable organism, Saccharomyces cerevisiae, in an effort to elucidate the mechanism of E4orf4-mediated cell death. E4orf4 expression is lethal in yeast cells and this toxicity is dependent on E4orf4 having a functional interaction with Cdc55, the yeast homolog of Balpha. Through its interaction with the B regulatory subunit, which determines substrate specificity, E4orf4 may inhibit or promote the dephosphorylation of selected PP2A substrates. / E4orf4 expression can induce high levels Clb2-Cdc28 activity and mitotic arrest in a Cdc55-dependent manner. Since E4orf4 targets only the Cdc55-containing pool of PP2A, the E4orf4-induced mitotic arrest suggests that PP2A-Cdc55 plays a direct role in regulating exit from mitosis. Two anaphase-promoting complexes (APCs) control mitotic exit, APCCdc20 and APCHct1. We find that E4orf4 induces premature APCCdc20 activity resulting in the premature degradation of Pds1 and Scc1 in a Cdc55-dependent manner. In contrast, E4orf4 did not induce APCHct1 as evidenced be the stability of its substrates, Cdc20, Clb2 and Cdc5 as well as the hyperphosphorylation of Hct1. E4orf4 prevents Cdc55-containing PP2A complexes from localizing normally, another mechanism by which E4orf4 may modulate PP2A activity towards substrates. We propose that E4orf4 promotes mitotic arrest by acting as a Cdc55-specific inhibitor of PP2A and that PP2A plays a role in controlling the timing of anaphase by regulating APCCdc20 activity.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause a chronic lung infection in individuals with cystic fibrosis. The O antigen of lipopolysaccharide is a major virulence factor for Gram-negative bacteria and plays a critical role in bacterium-host interactions. The inhibition of O antigen biosynthesis is known to significantly reduce the virulence of P. aeruginosa. The O antigen biosynthetic enzymes are considered as promising novel targets to develop new antimicrobials to treat a P. aeruginosa infection. / WbpP and WbpM are two UDP-GlcNAc-modifying enzymes that are involved in the initial steps of the O-antigen biosynthesis in P. aeruginosa serotype 06. WbpP is a UDP-GlcNAc 4-epimerase that preferentially catalyzes the conversion of acetylated substrates over non-acetylated substrates, such as UDP-Glc. In contrast, WbpM is a membrane protein with a catalytic domain that is capable of UDP-GlcNAc 4,6-dehydratase activity. The structural and biological studies were pursued to gain insight into the mechanism of action for WbpP and WbpM. Specifically for WbpP, the crystal structures of this enzyme in complex with cofactor and either UDP-Glc or UDP-GalNAc were determined at 2.5 and 2.1 A, respectively. These represent the first structural studies of a genuine UDP-GlcNAc 4-epimerase. For WbpM, crystallographic studies proved difficult. Hence, the crystal structures of FlaA1, a close soluble relative from Helicobacter pylori, were determined in the presence of substrate, inhibitors and bound cofactor, with resolutions ranging from 2.8-1.9 A. These represent the first structural studies of a 4,6-dehydratase that can catalyze a UDP-hexose. Third, the crystal structures of the Y141M FlaA1 mutant were determined in oxidized and reduced forms at 2.2 and 2.1 A, respectively, to study the structure of an unusual T-M-K triad found in WbpM. Finally, a homology model of the catalytic domain of WbpM was built based on the structures of FlaA1 wild type and mutant to unveil its unique structural features. / In conclusion, the work described in this thesis gives insight into the structures and reaction mechanisms of WbpP and WbpM and provide a basis for future studies aimed at understanding the O antigen biosynthesis of P. aeruginosa.