The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1p/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we developed a robust affinity purification-mass spectrometry (AP-MS) technique to generate protein-protein interaction maps for the Ubl systems. Each bait was systematically expressed as a C-terminal HA-tagged fusion protein in S. cerevisiae. A standardized method in which affinity purification via the HA epitope, followed by mild washing and mass spectrometric analysis, was performed and the data generated were used to build interaction maps. Affinity purification of the Rub1p E3 ligase Dcn1p identified a novel interaction with the AAA ATPase Cdc48p. This interaction was further studied to determine its biological significance.

The ubiquitin-like proteins (Ubls) are small polypeptides that function as post-translational modifications. Modification of a protein with a Ubl can alter its localization, activity and/or half-life. SUMO and Rub1p/Nedd8 are two Ubls that play important roles in a number of critical cellular processes, yet their specific cellular functions remain poorly understood. To better understand these important Ubls, we developed a robust affinity purification-mass spectrometry (AP-MS) technique to generate protein-protein interaction maps for the Ubl systems. Each bait was systematically expressed as a C-terminal HA-tagged fusion protein in S. cerevisiae. A standardized method in which affinity purification via the HA epitope, followed by mild washing and mass spectrometric analysis, was performed and the data generated were used to build interaction maps. Affinity purification of the Rub1p E3 ligase Dcn1p identified a novel interaction with the AAA ATPase Cdc48p. This interaction was further studied to determine its biological significance.

No description available.

atfish production is the largest aquaculture industry in the United States and infectious agents are responsible for 45% of all economic losses. Ictalurid herpesvirus 1 or Channel catfish virus (CCV) has a great economic impact on channel catfish aquaculture; yet it also has the potential for becoming a highly efficient vaccine vector eliciting long-lived immune responses against itself and, as a recombinant, other important catfish pathogens (bacteria, myxosporean, and fungi). However, little is known about CCV’s genome, its gene functions or genetic interactions with its host. Better understanding of CCV biology and pathogenesis could enable more rational vaccine design and other control strategies for CCV. My thesis is that “systems biology” can enable much more rapid understanding of CCV biology and pathogenesis. To test this thesis I needed to first more fully annotate the CCV genome, then construct a rapid system for generating CCV mutants and recombinants for systems biology research and then apply these tools in a systems biology experiment. I experimentally annotated the CCV proteome by proteogenomic mapping followed by real-time PCR and confirmed the expression of 37 of the 76 previously predicted ORFs (25 for the first time) as well as 17 novel ORFs. I next constructed two different infectious clones of CCV: one as three overlapping bacterial artificial chromosomes (BACs) and the other as a full length CCV BAC. These CCV BACs facilitate CCV mutant and recombinant production and I regenerated a genotypically wild-type and an attenuated virus. To further simplify CCV mutant production, I next adapted the CCV infectious clone for lambda phage crossover recombination cloning to enable sequence transfer into a specific CCV locus by a simple one-step in-vitro reaction. Finally, I used the CCV infectious clone, in combination with affinity purification, to identify interacting partners of the CCV zinc RING finger proteins ORF9, ORF11 and ORF12 to provide insight into the topology of one presumptive CCV-channel catfish molecular interaction network module. The work in this dissertation supports my thesis and the CCV BAC tools were patented; together these provide tools to facilitate and accelerate the development and testing of better CCV vaccines.

The mutated forms of the Discs overgrown gene causes overproliferation of imaginal discs of Drosophila melanogaster. Somatic mutations in its human counterpart, casein kinase I epsilon, were strongly associated with human breast cancer. Using the advantage of a high conservancy between fly's dco and human casein kinase I epsilon genes we have chosen D. melanogaster as a model organism to provide a list of probable Dco interaction partners via tandem affinity purification and mass spectrometry analysis. However, these proteins need to be independently verified as true Dco interaction partners.

No description available.

Voltage-gated sodium channels auxiliary subunits evolutionary emerged nearly 500 million years ago during the Cambrian explosion. These subunits alter one the most important ion channels to electrical signaling, the voltage-gated sodium channels support the propagation of electric impulses in animals. The mechanism for the auxiliary subunits effects on the channels is poorly understand, as is the stoichiometry between the auxiliary subunit and the channel. The focus of my thesis is to generate assays and to use these approaches to understand the interactions different types of voltage-gated channels and their auxiliary subunits. A biochemical approach was taken to identify novel interactions between the eukaryotic sodium channel auxiliary subunits and a prokaryotic voltage-gated sodium channel, a protein that diverged from the eukaryotic voltage-gated sodium channels billions of years ago. These interactions between the auxiliary subunits and channels were probed with chemical and photochemical crosslinkers in search of interaction surfaces and similarity to explain the mechanisms of interaction. The work in this thesis identified novel interactions between the voltage-gated sodium channel auxiliary subunits and voltage-gated channels that are distantly related to the voltage-gated sodium channels principally thought to be modulated by the auxiliary subunits. From this work a rudimentary concept can be theorized that the voltage-gated sodium channel β-subunits and not only β1 have a more primary role in electrophysiology by associating with multiple different types of ion channels.

The molecular chaperones are a group of proteins that are effective in vitro and in vivo folding aids and show a well documented affinity for proteins lacking tertiary structure. Heat-induced Escherichia coli BL21 cell lysate (10 mg protein) was applied to immobilized ɑ-casein (45 mg/g beads) or β-casein (30 mg/g beads) column. After removing a majority of nonspecifically bound proteins with 1 M NaCl, the molecular chaperones were eluted with cold water, 1 mM Mg-ATP, or 6 M urea. Western analysis identified five Escherichia coli molecular chaperones including DnaK, DnaJ, GrpE, GroEL, and GroES. Among samples, ATP eluates showed the highest chaperone purity of 80-87% followed by cold water eluates with 62-68% purity. The β-Casein column showed a higher binding capacity than the ɑ-casein column since β-casein urea eluates contained 3.18 mg total protein (or 58% chaperone) compared to a-casein urea eluates with 2.68 mg total protein (or 32% chaperone). For strain comparison, Escherichia coli NM522 eluates showed more unidentified proteins in cold water eluates from both affinity columns. Chaperones were induced from BL21 strain with three treatments: heat shock at 39°C, heat shock at 42°C, and alcohol shock with 3% ethanol (v/v). Lysates were applied to an immobilized β-casein (30 mg/g beads) column. The molecular chaperones were eluted with cold water or 1 mM Mg-ATP after washing with 1 M NaCl. The purity of eluted chaperones was 58% with cold water and 100% with Mg-ATP. The treatment at 42°C was the most efficient for chaperone induction with highest chaperone yield of 1.0 mg among samples. Refolding denatured carbonic anhydrase B enzyme in the presence of Mg-ATP resulted in a 97% recovery of heat-denatured enzyme and a 68% recovery of chemically denatured enzyme. It was concluded that the novel casein affinity chromatography is a rapid and efficient method for purification of chaperone. The affinity purified chaperones were effective in vitro folding aids.

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.

N-methyl D-aspartate (NMDA) receptors represent integral signal transducers for excitatory glutamate neurotransmission. While NMDA receptors are critical for synaptic plasticity, the molecular events underlying this process are not fully elucidated. The potential role of NP22, a novel neuronal protein, as a downstream mediator of NMDA receptor function is explored. NP22 protein expression in genetic and pharmacological models of NMDA receptor hypofunction is examined and no significant changes are reported. Characterization of the NP22 protein complex via tandem-affinity and FLAG-purification coupled with mass spectrometry was used and no novel protein interactions are reported. GFP-tagged NP22 colocalization with F-actin decreases in cell processes of transiently transfected HEK293 cells in response to elevated intracellular calcium, while similar colocalization reductions are not seen in stably transfected HEK293 under a comparable treatment regiment. Changes in intracellular calcium affecting NP22 biology can be useful in the ongoing characterization of this novel protein.