Exploring the interplay of structure, stability, activity and localization in tRNA nucleotidyltransferase function

The enzyme tRNA nucleotidyltransferase is of central importance in eukaryotic cells since it is required in multiple intracellular locations (nucleus, cytosol, mitochondria and plastids) for the production of mature tRNAs needed for protein synthesis. Therefore, tRNA nucleotidyltransferase serves as an excellent model protein to explore the complex roles that localization, activity, structure and stability play in defining protein function. I investigated how changes in the amino acid sequence of tRNA nucleotidyltransferase affect its structure, stability, activity and localization and may be linked, ultimately, to human disease. Using Arabidopsis thaliana as a model organism, I was the first to show that amino acid substitutions located within a region of tRNA nucleotidyltransferase required for enzyme activity, but outside of the enzyme’s classical amino-terminal organellar targeting sequence, affect localization to plastids and mitochondria. These studies suggest a direct link between intracellular distribution and how tightly the protein has folded. Moreover, these data indicate that the same seven amino acid changes that result in altered stability also reduce tRNA binding and enzyme activity. To more clearly define the role that these amino acids have in enzyme structure and function and to determine whether changes in protein stability, substrate binding, or structure bring about altered localization, I have fine-structure mapped the region encompassing residues 399-420 and shown that a single amino acid substitution (K418E) can dramatically alter tRNA binding and enzyme activity without detectable effects on enzyme structure. Localization studies are now underway to determine whether the K418E substitution, or other single substitutions within this region, alters the intracellular localization of tRNA nucleotidyltransferase. Using a second model organism, Saccharomyces cerevisiae, I have further explored the link between stability and activity in protein function. I showed that a single amino acid substitution (E189F) resulted both in a 5ºC decrease in thermal stability and a 25-fold decrease in enzyme activity and caused a temperature-sensitive phenotype. Moreover, converting arginine 64 to tryptophan in the variant enzyme restored enzyme activity and suppressed the temperature-sensitive phenotype, but did not restore thermal stability. These data suggest that the temperature-sensitive phenotype is defined by the reduction in activity and not thermal stability. Moreover, these data were the first to suggest a role for conserved motif C in active site organization and tRNA nucleotidyltransferase function. Finally, I applied the techniques and insight acquired from my studies of the Arabidopsis and yeast enzymes to explore how seven distinct amino-acid substitutions in human tRNA nucleotidyltransferase, all previously identified in patients suffering from sideroblastic anemia with B cell immunodeficiency, periodic fevers and development delay (SIFD), affect enzyme structure, stability and function in vitro. I showed that each of these mutations affects some combination of tRNA nucleotidyltransferase structure, activity, or stability. As expected, substitutions (T154I, M158V, L166S, R190I and I223T) within the conserved catalytic amino terminal region of the protein altered stability and/or activity but more interestingly two substitutions (I326T and K416E) in the less well conserved carboxy-terminal region of the enzyme altered catalytic efficiency, tRNA binding and quaternary structure. These studies have shown that a protein’s function can be defined by a combination of some or all of its structure, stability, activity or localization

Digital Genome-Wide ncRNA Expression, Including SnoRNAs, across 11 Human Tissues Using PolyA-Neutral Amplification

Non-coding RNAs (ncRNAs) are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6) is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I) cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available

Contrasting Patterns of Transposable Element Insertions in Drosophila Heat-Shock Promoters

The proximal promoter regions of heat-shock genes harbor a remarkable number of P transposable element (TE) insertions relative to both positive and negative control proximal promoter regions in natural populations of Drosophila melanogaster. We have screened the sequenced genomes of 12 species of Drosophila to test whether this pattern is unique to these populations. In the 12 species' genomes, transposable element insertions are no more abundant in promoter regions of single-copy heat-shock genes than in promoters with similar or dissimilar architecture. Also, insertions appear randomly distributed across the promoter region, whereas insertions clustered near the transcription start site in promoters of single-copy heat-shock genes in D. melanogaster natural populations. Hsp70 promoters exhibit more TE insertions per promoter than all other genesets in the 12 species, similarly to in natural populations of D. melanogaster. Insertions in the Hsp70 promoter region, however, cluster away from the transcription start site in the 12 species, but near it in natural populations of D. melanogaster. These results suggest that D. melanogaster heat-shock promoters are unique in terms of their interaction with transposable elements, and confirm that Hsp70 promoters are distinctive in TE insertions across Drosophila

Targeting the IGF-Axis in Cultured Pediatric High-Grade Glioma Cells Inhibits Cell Cycle Progression and Survival

Pediatric high-grade gliomas (pHGG) accounts for approximately 8–12% of primary brain tumors in children. Prognosis is poor, with a median survival of 9–15 months. Insulin-like growth factor 1-receptor (IGF-1R) gene amplifications have been identified in high-grade gliomas and may contribute to its highly aggressive phenotype, but the effect of IGF inhibitors on pHGG is yet to be determined. In the present study, we analyzed the response of patient-derived pediatric high-grade glioma cells to a novel IGF-1R inhibitor, the IGF-Trap. Using immunohistochemistry, we found that IGF-1R was localized to both the nucleus and cell membrane in different pHGG patient-derived xenograft (PDX) lines under basal conditions. In response to ligand binding, nuclear levels of the receptor increased, and this was associated with the transcriptional upregulation of both the receptor and cyclin D1, suggesting that IGF-1R could regulate its own expression and cell cycle progression in these cells. Insulin-like growth factor-1 (IGF-1) increased the proliferation of the pHGG cells DIPG13 and SGJ2, and this could be blocked by the addition of the IGF-Trap. The IGF-Trap reduced the colony formation of these cells in an optimal growth medium and impeded the ability of IGF-1 to rescue DIPG13 cells from starvation-induced apoptosis. Collectively, these results implicate the IGF-1 axis in the regulation of cell cycle progression, cellular proliferation, and cell survival in pHGG, and identify the IGF-axis as a target and the IGF-Trap as a potential inhibitor of this axis in pHGG

Herpesvirus trigger accelerates neuroinflammation in a nonhuman primate model of multiple sclerosis.

Pathogens, particularly human herpesviruses (HHVs), are implicated as triggers of disease onset/progression in multiple sclerosis (MS) and other neuroinflammatory disorders. However, the time between viral acquisition in childhood and disease onset in adulthood complicates the study of this association. Using nonhuman primates, we demonstrate that intranasal inoculations with HHV-6A and HHV-6B accelerate an MS-like neuroinflammatory disease, experimental autoimmune encephalomyelitis (EAE). Although animals inoculated intranasally with HHV-6 (virus/EAE marmosets) were asymptomatic, they exhibited significantly accelerated clinical EAE compared with control animals. Expansion of a proinflammatory CD8 subset correlated with post-EAE survival in virus/EAE marmosets, suggesting that a peripheral (viral?) antigen-driven expansion may have occurred post-EAE induction. HHV-6 viral antigen in virus/EAE marmosets was markedly elevated and concentrated in brain lesions, similar to previously reported localizations of HHV-6 in MS brain lesions. Collectively, we demonstrate that asymptomatic intranasal viral acquisition accelerates subsequent neuroinflammation in a nonhuman primate model of MS

Spatiotemporal distribution of fibrinogen in marmoset and human inflammatory demyelination.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system. Although it has been extensively studied, the proximate trigger of the immune response remains uncertain. Experimental autoimmune encephalomyelitis in the common marmoset recapitulates many radiological and pathological features of focal multiple sclerosis lesions in the cerebral white matter, unlike traditional experimental autoimmune encephalomyelitis in rodents. This provides an opportunity to investigate how lesions form as well as the relative timing of factors involved in lesion pathogenesis, especially during early stages of the disease. We used MRI to track experimental autoimmune encephalomyelitis lesions in vivo to determine their age, stage of development, and location, and we assessed the corresponding histopathology post-mortem. We focused on the plasma protein fibrinogen-a marker for blood-brain barrier leakage that has also been linked to a pathogenic role in inflammatory demyelinating lesion development. We show that fibrinogen has a specific spatiotemporal deposition pattern, apparently deriving from the central vein in early experimental autoimmune encephalomyelitis lesionsold, and preceding both demyelination and visible gadolinium enhancement on MRI. Thus, fibrinogen leakage is one of the earliest detectable events in lesion pathogenesis. In slightly older lesions, fibrinogen is found inside microglia/macrophages, suggesting rapid phagocytosis. Quantification demonstrates positive correlation of fibrinogen deposition with accumulation of inflammatory cells, including microglia/macrophages and T cells. The peak of fibrinogen deposition coincides with the onset of demyelination and axonal loss. In samples from chronic multiple sclerosis cases, fibrinogen was found at the edge of chronic active lesions, which have ongoing demyelination and inflammation, but not in inactive lesions, suggesting that fibrinogen may play a role in sustained inflammation even in the chronic setting. In summary, our data support the notion that fibrinogen is a key player in the early pathogenesis, as well as sustained inflammation, of inflammatory demyelinating lesions

Spatiotemporal distribution of fibrinogen in marmoset and human inflammatory demyelination.

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system. Although it has been extensively studied, the proximate trigger of the immune response remains uncertain. Experimental autoimmune encephalomyelitis in the common marmoset recapitulates many radiological and pathological features of focal multiple sclerosis lesions in the cerebral white matter, unlike traditional experimental autoimmune encephalomyelitis in rodents. This provides an opportunity to investigate how lesions form as well as the relative timing of factors involved in lesion pathogenesis, especially during early stages of the disease. We used MRI to track experimental autoimmune encephalomyelitis lesions in vivo to determine their age, stage of development, and location, and we assessed the corresponding histopathology post-mortem. We focused on the plasma protein fibrinogen-a marker for blood-brain barrier leakage that has also been linked to a pathogenic role in inflammatory demyelinating lesion development. We show that fibrinogen has a specific spatiotemporal deposition pattern, apparently deriving from the central vein in early experimental autoimmune encephalomyelitis lesionsold, and preceding both demyelination and visible gadolinium enhancement on MRI. Thus, fibrinogen leakage is one of the earliest detectable events in lesion pathogenesis. In slightly older lesions, fibrinogen is found inside microglia/macrophages, suggesting rapid phagocytosis. Quantification demonstrates positive correlation of fibrinogen deposition with accumulation of inflammatory cells, including microglia/macrophages and T cells. The peak of fibrinogen deposition coincides with the onset of demyelination and axonal loss. In samples from chronic multiple sclerosis cases, fibrinogen was found at the edge of chronic active lesions, which have ongoing demyelination and inflammation, but not in inactive lesions, suggesting that fibrinogen may play a role in sustained inflammation even in the chronic setting. In summary, our data support the notion that fibrinogen is a key player in the early pathogenesis, as well as sustained inflammation, of inflammatory demyelinating lesions