Browsing by Author "York, John D"

Bisphosphate 3'-nucleotidase (Bpnt1) is a member of a family of small molecule phosphatases whose activities depend on divalent cations and are inhibited by lithium. While the enzymes share many commonalities, they have distinct and non-­overlapping substrate pools. Of the seven mammalian members, two enzymes, gPAPP and Bpnt1, hydrolyze the same small molecule 3'-phosphoadenosine 5'-phosphate (PAP) but act in separate subcellular compartments, the Golgi apparatus and cytoplasm respectively. Hydrolysis of PAP, which is a metabolite of the inorganic sulfate incorporation pathway, is highly conserved throughout evolution from bacteria to yeast to humans. Evidence in multiple species has shown that inhibiting PAP hydrolysis leads to cellular toxicity as a result of its accumulation and also that these effects can be ameliorated by modulating the rate of its production. However, despite the abundant evidence of its importance

from studies in lower eukaryotes, the role of the cytoplasmic PAP phosphatase, Bpnt1, in more complicated mammalian physiological remains poorly understood. Here we report for the first time the generation and characterization of mice deficient for Bpnt1. Bpnt1 null mice do not exhibit skeletal defects, but instead develop severe liver

pathologies and deficiencies in intestinal iron absorption. Loss of Bpnt1 leads to tissue-specific elevations of the substrate PAP. To test the hypothesis that a toxic cellular accumulation of PAP accounts for the observed phenotypes, we generated a double mutant mouse that concomitantly down regulates bisphosphorylated nucleotide synthesis in the context of Bpnt1 deficiency. Remarkably, double mutants do not display any detectable physiological defects seen in Bpnt1 null mice. In addition, we have identified and characterized a novel substrate of 3'­nucleotidases, 3'-phosphoadenosine 5'-diphosphate (PAPP) that co-accumulates with PAPS and PAP and might play a role in mediating certain aspects of the physiological defects of Bpnt1 null mice. Overall, our study defines a role for Bpnt1 in mammalian physiology and provides mechanistic insights into the importance of cytoplasmic 3'-­

nucleotide hydrolysis to normal cellular function.

Inositol phosphates (IPs) are versatile metabolites that play important roles in multiple cellular processes. They have been considered signaling messengers that relay extracellular signals via a wave of their production and allosteric regulation of downstream targets. In addition to this classical role, recent studies have revealed that certain IPs can also function as protein structural cofactors. However, except for the two plant hormone receptors TIR1 and COI1, these IP binding proteins have neither sequences nor functions in common. Therefore, to test whether other cellular proteins are also subjected to this type of regulation and whether an IP binding motif exists, more proteins that bind IPs in a similar manner need to be identified. Via a proteome-wide biochemical screen, two yeast proteins were found to contain IP₆ as an integral component. One is the N-terminal acetyltransferase A complex (NatA), and the other one is Tif31 (or Clu1). IP₆ binding was also observed in NatC, another N-terminal acetyltransferase. The bioinformatics analysis and mutagenesis study showed that tandem tetratricopeptide repeats (TPRs), the only common structural element of NatA and Tif31, were responsible for coordinating IP₆. This mechanism of IP_{6 binding is conserved in the fly homologs of these proteins.}

NatA is one of the enzymes that acetylate the α-amino groups at protein N-termini. This widespread protein modification affects a wide range of cellular processes. IP₆ was shown to be essential for yeast NatA in vitro thermostability and for some but not all functions of the protein in cells grown under temperature stress. Other multiple phosphate-containing molecules including IP₅ species and the bacterial alarmone ppGpp were found to bind NatA and partially compensate for the lack of IP₆. IP₆ also binds the human NatA homolog. This binding is crucial for hNatA complex formation, in vitro and in vivo activities, and ability to rescue NatA-deficient phenotypes when it is expressed in yeast. Therefore, IP₆ acts as a molecular glue that brings hNatA (and hNatE) subunits together. The other protein found in our screen, Tif31, is important for normal mitochondrial morphology and distribution. In cells that cannot produce IP₄, IP₅ and IP₆, Tif31 levels were significantly decreased and these cells exhibited severe mitochondrial aggregation. Tif31 mutants that cannot bind IP₆ showed a reduction in cellular levels, a shift to high molecular weight complexes or aggregates, and inability to rescue tif31δ mitochondrial phenotype. This study established the vital role of IP₆ and IP₅ in maintaining Tif31 stability and Tif31-mediated regulation of mitochondrial distribution.

Collectively, this dissertation discovered two proteins that use IP₆ as a structural cofactor. For the first time, a conserved IP₆ binding motif has been shown to be present in certain TPR-containing proteins. Via tight binding to these proteins, IP₆ stabilizes their structures or subunit interaction. This research provides mechanistic evidence for the interplay between IP biology and N-terminal acetylation as well as between IP biology and mitochondrial morphology.

Inositol phosphates (IPs) are eukaryotic signaling molecules that play important roles in a wide range of biological processes. IPs are required for embryonic development and patterning, insulin secretion, the regulation of telomere length, proper progression through the cell cycle, and the regulation of ion channels. This work uses the yeast Saccharomyces cerevisiae as a model system for investigating the functions of IPs and focuses on the transcriptional regulation of the gene encoding the secreted mating pheromone MFα2 by the IP kinase Ipk2 (also called Arg82, ArgR3, and IPMK). This work shows that Ipk2 has both kinase-dependent and kinase-independent functions in regulating the transcription of MFα2. Transcription of MFα2 is also dependent upon the integrity of an Mcm1-binding site in its promoter. This is the first description of a role for this binding site in the transcription of MFα2.

In vivo and in vitro screening approaches to identify additional factors associated with MFα2 expression or with IP biology generally are also described. These unbiased approaches provide some valuable insight for further investigations.

NatA binds inositol hexakisphosphate and other ligands, and exhibits conformational flexibility dependent on the ligand bound.

Lipopolysaccharide, the physical barrier that protects Gram-negative bacteria from various antibiotics and environmental stressors, is anchored to the outer membrane by the phosphorylated, acylated disaccharide of glucosamine known as lipid A. Besides being necessary for the viability of most Gram-negative bacteria, lipid A interacts directly with specific mammalian immune cell receptors, causing an inflammatory response that can result in septic shock. The lipid A biosynthetic pathway contains nine enzymatic steps, the sixth being the phosphorylation of the tetraacyldisaccharide-1-phosphate (DSMP) precursor to form lipid IV_A by the inner membrane-bound kinase LpxK, a divergent member of the P-loop containing nucleotide triphosphate hydrolase superfamily. LpxK is the only known P-loop kinase to act on a lipid at the membrane interface.

We report herein multiple crystal structures of Aquifex aeolicus LpxK in apo as well as ATP, ADP/Mg2+, AMP-PCP, and chloride-bound forms. LpxK consists of two α/β/α sandwich domains connected by a two-stranded β-sheet linker. The N-terminal domain, which has most structural homology to other P-loop kinase family members, is responsible for catalysis at the P-loop and positioning of the DSMP substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate using a 25º hinge motion about its base which also assembles the necessary catalytic residues at the active site.

Using a thin-layer chromatography-based radioassay, we have performed extensive kinetic characterization of the enzyme and demonstrate that LpxK activity in vitro is dependent on the presence of detergent micelles, the use of divalent cations, and formation of a ternary LpxK-ATP/Mg2+-DSMP complex. Implementing steady-state kinetic analysis of multiple point mutants, we identify crucial active site residues. We propose that the interaction of D99 with H261 acts to increase the pK_a of the imidazole group, which in turn serves as the catalytic base to deprotonate the 4’-hydroxyl of DSMP. An analogous mechanism has not yet been reported for any member of the P-loop kinase family.

The membrane/lipid binding characteristics of LpxK have also been also investigated through a crystal structure of the LpxK-lipid IV_A product complex along with point mutagenesis of residues in the DSMP binding pocket. Critical contacts with the bound lipid include interactions along the glucosamine backbone and the 1-position phosphate group, especially through R171. Furthermore, analysis of truncation mutants of the N-terminal helix of LpxK demonstrates that this substructure is a critical hydrophobic contact point with the membrane, and that both charge-charge and hydrophobic interactions contribute to the localization of LpxK at the lipid bilayer.

Overall, this work has contributed significantly to the limited knowledge surrounding membrane-bound enzymes that act upon lipid substrates. It has also provided insight into the process of enzyme evolution as LpxK, while containing a similar core domain as other P-loop kinases, has developed multiple subdomains required for both cellular localization and recognition of novel substrates. Finally, the presence of multiple crystal structures and detailed understanding of the LpxK catalytic mechanism will improve the chances of successfully targeting this essential step in lipid A biosynthesis in the pursuit of novel antimicrobials.

Inositol Polyphosphate Multi-Kinase (IPMK, also known as ArgRIII, Arg82, and IPK2) is a central component of the inositol signaling system, catalyzing the phosphorylation of at least four different inositol polyphosphate species in vivo with in vitro activity observed for three more. Each of these IP species is sterically unique and the phosphorylation target varies between the 6'-, 3'-, or 5'-hydroxyls, classifying IPMK as a 6/3/5-kinase. The products of IPMK have been linked to multiple processes including cell cycle regulation, transcriptional control, telomere length regulation, mRNA export and various phenotypes including mouse embryonic and fly larvae development, and stress responses in plants and yeast. Linking specific IP species and cellular processes has been complicated by the inability to distinguish between the different effects of the various IP species generated by IPMK. Deletion of IPMK affects the IP populations of all its various substrates and products and therefore the role of a single IP species cannot be tracked. The goals of this work were to address the question of substrate selectivity and develop new tools to probe inositol signaling in vivo through a combination of structural, enzymatic, and genomic techniques.

The structure of Arabidopsis thaliana IPMK is reported at 2.9Å resolution and in conjunction with a new model of inositol selectivity has been used to design constructs with altered substrate profiles. In vitro and in vivo experiments have confirmed that IPMK identifies substrate inositol polyphosphate species through a recognition surface that requires phosphate groups occupy specific pockets and rejects those with axial phosphate groups in specific regions. In vivo experiments have linked specific inositol polyphosphate species to nitrogen metabolism and temperature sensitivity in yeast and established the potential for these constructs to be used to probe signaling in other organisms.