Master of Science / Department of Biochemistry / Gerald R. Reeck / Armet is a bifunctional protein widely distributed in animal species, vertebrate and invertebrate. It is an evidently part of the Unfolded Protein Response (UPR) and promotes survival in cells that are under endoplasmic-reticulum (ER) stress. It has also been found as a secreted protein with neurotrophic activity. The crystal and solution structures of human Armet show it is a helix-rich protein with two domains linked through a flexible linker region. In this study, immunofluorescence staining was used to verify Armet’s localization in ER and Golgi apparatus in MBA-MD-231 cells. Evidence for calcium binding by Armet was obtained by circular dichroism spectroscopy (the binding of calcium appeared to decrease helix content), by differential scanning calorimetry (binding of calcium resulted in a less structured protein) and two-dimensional (1H-15N HSQC) nuclear magnetic resonance spectroscopy. A difference HSQC spectrum of Armet, with and without calcium, showed peaks of increased intensity, of decreased intensity and of perturbed chemical shift. There were about 30 such peaks in total. Several of these affected amino acid residues appeared to form a cluster of negatively charged side chains that could possibly form a binding site for a calcium ion. Heterogeneity of three types was observed in recombinant Armet expressed in E. coli cells. Two bands of slightly different mobility were observed in SDS gels run in the absence of reducing agent. These may represent alternate arrangements of disulfide bonds, as previously reported by other investigators but not explained. Further, in the absence of reducing agent, a faint ladder was formed by human Armet, indicating formation of disulfides between Armet molecules. Oligomers with sedimentation coefficient greater than the monomeric protein, in the absence of reducing agent, disappeared in the presence of a reducing agent. Finally, minor species of mass differences of 98 and 180 with respect to the main protein component were observed by MALDI-TOF mass spectrometry. These studies provide a more thorough characterization of Armet than has been previously available and set the scene for future investigations of the binding of organic ligands to the protein.

Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald R. Reeck / Armet has been found in mammalian systems to be a bi-functional protein that is secreted extracellularly and is also found in the endoplasmic reticulum. It has been shown to be a neurotrophic factor and also a member of the unfolded protein response. Transcript knockdown of Armet via RNA interference in late instar larvae of Tribolium castaneum produces a fatal phenotype during eclosion from pupa to adult. Initial observations of pupae cuticle indicate disorganization of cuticles in insects with the Armet transcript knocked down. Here I expand studies on the effects of dsArmet RNA injection; both in a wild type strain and a fluorescent strain of Tribolium, and discuss possible mechanisms for the fatal phenotype.

Doctor of Philosophy / Biochemistry and Molecular Biophysics Interdepartmental Program / Gerald R. Reeck / Armet is a bifunctional protein that is apparently universally distributed among multicellular animal species, vertebrate and invertebrate alike. A member of the Unfolded Protein Response, (UPR) Armet promotes survival in cells that are under endoplasmic-reticulum (ER) stress. I have carried out biophysical studies on human Armet looking for compounds that bind to Armet and hence could reduce its anti-apoptotic function, thus potentially joining the growing class of pro-apoptotic drugs. Performed primarily with 1H-15N HSQC NMR, ligand studies showed that approximately 60 of the 158 residues are potentially involved with binding. The 60 residues are distributed throughout both domains and the linker suggesting multi-domain interaction with the ligand. Circular dichroism studies showed heat denaturation in a two-step unfolding process with independent unfolding of both domains of Armet with Tm values near 68°C and 83 C with the C-terminal domain unfolding first, as verified by 1H-15N HSQC NMR measurements. I also provide the first identification of UPR transcripts in pea aphids, Acyrthosiphon pisum, the genetic model among aphids. I measured transcript abundance with hope of finding future transcriptional targets for pest mitigation. I identified 74 putative pea aphid UPR components, and all but three of the components have higher transcript levels in aphids feeding on plants than those that fed on diets. This activated UPR state is attributed to the need for saliva proteins for plant feeding. Because aphids are agriculturally significant pests, and saliva is pivotal to their feeding on host plants, genes that encode saliva proteins may be targets for pest mitigation. Here I have sought the aphid’s saliva proteome by combining results obtained in several laboratories by proteomic and transcriptomic approaches on several aphid species. With these data I constructed a tentative saliva proteome for the pea aphid by compiling, collating, and annotating the data from several laboratories. I used RNA-seq to verify the transcripts in pea aphid salivary glands, thus expanding the proposed saliva proteome from approximately 50 components to around 130 components, I found that transcripts of saliva proteins are upregulated during plant feeding compared to diet feeding.

Master of Science / Department of Biochemistry / Gerald Reeck / Aphids mount a remarkable salivary secretion to overcome plant host defenses. Our group has previously reported a gene unique to aphids enriched in the salivary glands of the pea aphid A. pisum, C002, which is required for successful feeding on its host plant Vicia fava. Here I present an analysis of genetic variation within the available EST data for C002 in pea aphids. From 596 total ESTs, 332 are full-length, and segregate into 8 validated haplotypes based on the criteria I set in place to access the quality of EST data. Additionally, Armet, is a putative multi-functional gene implicated as a neurotrophic factor during development, and as a part of the unfolded protein response during stress. I employ RNA interference in the model organism T. castaneum to determine the effect of transcript knockdown during development from early in-star larval stages, through pupation, and its effect on adult emergence. I report that knockdown of Armet transcript significantly hinders the ability for beetles to emerge from the pupae.