Higuchi Biosciences Center - University of Kansas

Fall 2006 Science Talks
November 30, 2006

Abstracts 1-15 | Abstracts 16-30 | Abstracts 31-45 | Abstracts 46-54

31. Assay Development for Identification of Binding Partners to Phosphatase of Regenerating Liver (PRL-1) Protein Using Blue Native Gel Electrophoresis
Matthew L. Moreno, Jennifer Laurence
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS

The goal of the research project is to develop methods to successfully find binding partners associated with phosphatase of regenerating liver-1 (PRL-1) protein. PRL-1 has been shown to promote cell migration, invasion, and metastasis when expressed at elevated levels. The technique being utilized is Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE). BN-PAGE permits a high-resolution separation of multi-protein complexes under native (non-denaturing) conditions. The hydrophobic interaction between these complexes and the blue dye (Coomassie G-250), which has a negative charge, allows the complexes to migrate on the gel. BN-Agarose Gel Electrophoresis (BN-AGE), a variation of this technique, is also utilized for higher molecular weight protein complexes. In the future, BN-PAGE/BN-AGE western blots of cell lysates will be used to find additional binding partners. A substrate trapping mutant form of the protein was analyzed in vitro with viable binding partners. Preliminary results show nuclear localization of the protein through western blots from whole cell lysates as well as nuclear extracts. Previous studies have shown that PRL-1 has a high binding affinity for microtubulin, in particular, alpha and gamma tubulin. In the future, further methods will be developed to optimize the use of these techniques to conclusively identify the binding partners for PRL-1.

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32. Development of a Microdialysis-Microschip CE System for On-Line Monitoring
Pradyot Nandi, Nobuyuki Suzuki, Susan M. Lunte
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS
R. N. Adams Institute of Bioanalytical Chemistry, University of Kansas, Lawrence, KS

Purpose. Development of an online microdialysis-microchip capillary electrophoresis system for in-vivo peptide monitoring. Methods. A glass-PDMS hybrid device with a 2.5 cm separation channel was utilized as the microchip for on-chip derivatization of peptides and amino acids with naphthalene 2,3 dicarboxaldehyde/2-mercaptoethanol and subsequent detection by laser induced fluorescence (LIF). In-vivo microdialysis sampling of fluorescein from rat striatum was performed on-chip to evaluate the injection characteristics.
Results. Detection of peptides and amino acids were achieved following derivatization on-chip. In optimization studies using the MEKC buffer, temporal resolution of 10 s was achieved with 1 mM fluorescein when the analyte was sampled directly from the sample reservoir.
Conclusions. Further investigation regarding the effect of aCSF on voltage-gated injections will be performed based on the in-vivo experiments. A pneumatic valve based microchip with pressure actuated injection will be explored as an alternative to microchips with voltage based injection.

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33. Synthesis of PLGA Nanoparticles with Conjugated CLABL as Targeted Vascular Delivery Vehicles
Na Zhang1, Cory Berkland1,2
1Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS
2Department of Chemical and Petroleum Engineering, University of Kansas, Lawrence, KS

Introduction A variety of drug treatments have been investigated in recent decades outlining the potential of targeting specific cytokines or cell adhesion molecules. In particular, mAbs can target and bind molecules to disrupt the inflammatory pathway; however, this approach does not allow exclusive disruption of only those cells involved in the localized pathogenesis of the disease. As an alternative, nanoparticles may be targeted to ICAM-1, which is drastically up-regulated on the vascular endothelium local to increased production of pro-inflammatory cytokines such as interferon-gamma (IFN-γ), interleukin-1-beta (IL-1-β), tumor necrosis factor-alpha (TNF-α), etc. Our collaborator, Dr. Teruna Siahaan, has developed a peptide, cLABL (Cyclo(1,12)PenITDGEATDSGC), derived from Leukocyte Function-Associated Factor-1 that binds ICAM-1 with high avidity specificity as an alternative to expensive and immunologically active mAbs. The objective of this work is to evaluate nanoparticle targeting medicated by cLABL binding ICAM-1 for the purpose of targeted intracellular drug therapy. Experimental Methods PLGA nanoparticles were prepared using emulsification-solvent evaporation methods and solvent diffusion methods, respectively. Nanoparticles were designed to exhibit a high surface density of carboxyl groups by utilizing poly(ethylene-alt-maleic acid) as a surfactant. The carboxylic acid end group of PLGA nanoparticles is conjugated to the N-terminal of a PEG-spacer. Then the PEG-spacer is conjugated to cLABL peptide to give the desired PLGA-PEG-cLABL. The size and zeta potential of these nanoparticles were analyzed by dynamic Light scattering and phase analysis light scattering. Human umbilical vein endothelial cells (HUVECs) were studied to validate the effectiveness of nanoparticle targeting.
Results and Discussions The particle size of PLGA nanoparticles was between 180 to 360 nm for the different methods used. By carefully controlling reaction conditions, nanoparticles with conjugated cLABL could be synthesized with size distributions that closely match those suitable for IV administration particles. Fluorescence micrographs indicated rapid and sustained cell binding for cLABL nanoparticles, after incubated with FITC-Dextran, blank nanoparticles (NP), or cLABL-nanoparticles. Further work will be carried out to identificate the sub-cellular localization and the fate of the endocytosed biodegradable cLABL-nanoparticles.
Conclusion Nanoparticles coated with cLABL peptides are shown to preferentially pool to HUVECs exhibiting up-regulated ICAM-1.
Reference 1. Muro S, Gajewski C, Koval M, Muzykantov V.R. ,2005, ICAM-1 recycling in endothelial cells: a novel pathway for sustained intracellular delivery and prolonged effects of drugs, BLOOD, 15 (105):650-658

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34. Bile Salts Stimulate Recruitment of IpaB to the Shigella Surface Where it Colocalizes with IpaD at the Tip of the Type III Secretion Needle
AJ Olive, R Kenjale, ME Espina, ND Smith, D Moore, WL Picking, WD Picking
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

Shigella flexneri causes a severe form of dysentery that has worldwide public health implications. Shigella uses a type III secretion system (TTSS) to invade and deliver effector proteins into host cells to subvert normal cellular functions. Invasion plasmid antigen D (IpaD) is required for invasion because it controls the secretion and delivery of the IpaB and IpaC effector/translocator proteins into the host cell membrane. IpaD null mutants are noninvasive and constitutively secrete large amounts of other TTSS effector proteins. We have previously shown that IpaD localizes to the tip of the TTSS. Bile salts have been shown to increase adhesion and invasion of Shigella. In this study we found that incubating Shigella with deoxycholate (DOC) recruits IpaB to the surface where it co-localizes with IpaD at the needle tip. We also found, through fluorescence polarization experiments, that recombinant IpaD and IpaB interact in vitro suggesting these two proteins do interact at the tip of the TTSS needle. This is the first demonstration of a translocator being found at the needle tip, and we hypothesize that this formation of a secretion competent tip complex may allow for IpaB to serve as the host cell sensor.

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35. Transgenic Mice Over-expressing Glutamate Dehydrogenase 1 (GLUD1) in Neurons: A Model of Accelerated Loss of Neurons, Dendrites and Nerve Terminals Due to Aging
Ranu Pal, Xiaodong Bao, Xinkun Wang, Abdulbaki Agbas, S. Fowler, Elias K. Michaelis
Department of Pharmacology and Toxicology, Higuchi Biosciences Center, University of Kansas, Lawrence, KS

Aging is associated with modest losses of neurons and more substantial losses of dendrites and synapses in select brain regions, such as the hippocampus. Most neurons that suffer from age-associated damage use glutamate (Glu) as their neurotransmitter. Glu is the major excitatory transmitter in mammalian brain. Excessive extracellular accumulation of Glu, as may occur in aging, can lead to neuronal damage. Several enzymes are involved in the maintenance of Glu transmitter pools. We considered GLUD, a mitochondrial enzyme, to be a rate-limiting step in Glu transmitter biosynthesis. We created a transgenic (Tg) mouse model of hyper-GLUD activity by expressing the GLUD1 gene in neurons (under neuron-specific enolase promoter). At 5-8 mos of age, the GLUD1 Tg mice exhibited increased expression of GLUD1 protein and activity in brain neurons, and increased release of Glu in response to neuronal stimulation as compared with wild type (wt) littermates. At 14-16 mos, some Tg mice developed tremors/dyskinesias. The survival of Tg mice was reduced as compared with wt littermates. In brain and spinal cord, we have observed increased cell death with advancing age, but only in select brain regions, such as the CA1 field of the hippocampus (P < 0.05). In the same region, there was highly significant loss of dendrites that were labeled by antibodies to the microtubule-associated protein MAP2, as well as loss of nerve terminals labeled by synpaptophysin. A significant loss in MAP2 labeling was also observed in dendrites of the CA3 field of the hippocampus despite the very modest loss of neurons in that region. DNA microarray analyses of the hippocampus from 6-mo old Tg and wt mice revealed significant up-regulation of genes involved in dendrite and axon formation/growth in Tg mice. One such gene was that of the proline rich tyrosine kinase (PTK2B). This protein is distributed in dendrites and translocates to membranes upon activation (phosphorylation). Despite the loss of dendrites in Tg mice, there was a significant relative increase in PTK2B and phospho-PTK2B protein levels in dendrites of Tg mice as revealed by the increases in the ratio of PTK2B or phospho-PTK2B to MAP2 in the dendrites of CA1 neurons. Changes in these proteins with age are now being monitored as measures of the capacity of neurons to grow new dendrites following age-associated losses, possibly due to hyper-glutamatergic activity.
[This research was supported by grants from NIA, AG12993, and NICHD, HD02528, and a KTEC grant to the Higuchi Biosciences Center; the support of the Higuchi Biosciences Center, the University of Kansas, is also acknowledged.]

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36. Synthesis and Pharmacological Evaluation of Dual Labeled Delta Opioid Receptor Peptides
A.M. Peck1, V. Kumar2, T.F. Murray3, J.V. Aldrich1
1Department of Medicinal Chemistry, University of Kansas, Lawrence, KS
2Department of Pharmaceutical Sciences, University of Maryland, Baltimore, MD
3Department of Physiology and Pharmacology, College of Veterinary Medicine, University of Georgia, Athens, GA

Understanding receptor-ligand interactions is important in designing new receptor ligands. Opioid receptors, however, are difficult to study because they are trans-membrane receptors and have low expression levels in biological systems. We have previously prepared affinity labeled derivatives of opioid peptides in order to study their receptor-ligand interactions. Modified TIPP (Tyr-Tic-Phe-Phe, Tic = 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) derivatives containing a reactive functionality on Phe4 exhibit high affinity and wash-resistant inhibition of binding to delta opioid receptors [1,2]. Here we will describe dual labeled TIPP derivatives which contain a second functionality attached to the C-terminus via a hydrophilic linker to assist with detection and receptor isolation. We will present the synthesis of these modified TIPP derivatives and describe, for one analog, a side reaction that resulted in a novel cyclic peptide. Preliminary pharmacological data on the dual labeled TIPP derivatives will also be presented.

[1] Kumar, V., Murray, T.F., Aldrich, J.V. J. Med. Chem., 45, 3820 (2002).
[2] Maeda, D.Y., Berman, F., Murray, T.F., Aldrich, J.V. J. Med. Chem., 43, 5044 (2000).

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37. Surfactant Selection for the Development of “Nanoclusters” for Vaccine Delivery
Laura J. Peek, Lianjun Shi, Lydia M. Roberts, Cory Berkland
Chemical and Petroleum Engineering Department, University of Kansas, Lawrence, KS Pharmaceutical Chemistry Department, University of Kansas, Lawrence, KS

Introduction Pathogens such as viruses and bacteria are naturally particulate; therefore, the most effective vaccines often reflect this property. Exploiting this characteristic for enhancing vaccine efficacy makes developing vaccines especially challenging since particle size is difficult to control. Particles of approximately 2 μm in diameter have been shown to be efficiently delivered to the deep lung and taken up by antigen presenting cells. A vaccine for mucosal immunization delivered to the deep lung must utilize this particle size while delivering a large antigen payload. Antigens adsorbed to the surface of particles offer the advantage of improved antigen stability and activity as compared to encapsulated antigens; however, a major disadvantage is the reduced dose of antigen (for a constant particle mass) that can be delivered. Our hypothesis is that “nanoclusters” for pulmonary vaccine delivery will exhibit 100% fine particle fraction (aerodynamic diameter <5 μm) and adsorb >2% antigen by weight, similar to the nascent nanosuspension.
Experimental Methods Charged poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles (~200 nm) were prepared using an emulsion-solvent evaporation method and characterized by dynamic light scattering and zeta potential measurements. Cationic and anionic coating materials were utilized to modify the surface charge of the particles. Adsorption isotherms were generated using UV absorbance spectroscopy to quantify adsorption of a model protein antigen, ovalbumin, to the charged nanoparticles. Additionally, cationic and anionic nanoparticles were flocculated to form “nanoclusters.” The effect of particle concentration on the geometric and aerodynamic diameters was evaluated.
Results Adsorption of ovalbumin to charged PLGA nanoparticles seems to be governed primarily by electrostatic interactions although other forces may also have a role. Flocculation of nanoparticles is dependent on a variety of factors, including charge, concentration, vessel stir rate, etc.
Discussion The construction of adsorption isotherms for protein binding to charged, polymeric particles is a useful approach for determination of an appropriate coating material and particle size. These isotherms will be important for determining the processing conditions necessary for flocculation. Future investigations will focus on optimizing the nanoparticle and flocculate size to achieve maximum protein adsorption as well as characterizing the aerosol properties of the “nanoclusters.”
Conclusions Based on principles of simple electrostatics, the choice of an appropriate surfactant for particle fabrication can be guided by considering the isoelectric point of the protein antigen and pH conditions. Furthermore, “nanoclusters” can be fabricated using oppositely charged nanoparticles.

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38. Light as a Traceless Reagent for Maleimide Based Combinatorial Libraries: A New Application of the pHP Phototrigger
Chamani Perera, Richard S. Givens;
Department of Chemistry, University of Kansas, Lawrence, KS

Following the success of the p-hydroxyphenacyl (pHP) phototrigger as an efficient photoprotecting group for a variety of functional groups, its utilization in a traceless reagent release of combinatorial libraries is explored. Photoreleasable protecting groups has also received attention in solid phase organic synthesis due to ease of release and the potentially facile separation of polymer and product. Synthesis of a library of pHP caged maleimide derivatives of available amino acids (1) is in progress. Diels-Alder reactions with the maleimide dienophile provide additional diversification. For example, cyclopentadiene yields norbornene handles that can be used in ring opening metathesis polymerization (ROMP). The photorelease of 2 will also free a functional group (carboxylic acid) that can be used for further elaboration. The synthesis and the photolysis of the glycine and phenylalanine derivatives are being investigated.

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39. Stereoselective, De Novo Synthetic Route to a Combinatorial Library of Peptide-Linked Nucleosides
Wing (Kevin) C. Poon, Apurba Datta
Department of Medicinal Chemistry, University of Kansas, Lawrence, KS

In recent times, structurally modified nucleoside building blocks and oligonucleotides thereof have gained much attention by virtue of their significant biological profiles, including their use in antisense, antiviral, and anticancer therapies. This awareness of the potential therapeutic utility of nucleoside analogs has stimulated an intensive effort in the search for newer generations of structurally modified analogs designed to improve their potency and circumvent the various physical and biological limitations.

In the present research, a stereoselective, de novo synthetic route has been developed for the construction of a structurally unique class of C-4’ side chain modified peptide-linked nucleosides. The synthetic strategy and approach involves initial synthesis of a key chiral amino butenolide intermediate 1 followed by its efficient conversion to the desired peptidyl nucleosides 2. Employing the above route, and utilizing easily available nucleobases and amino acids as two diversity elements, synthesis of a twentyfour-membered combinatorial library of the title nucleosides have been accomplished. The details of the synthesis will be presented.

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40. Protein-excipient Interactions in Solid Protein Formulations
Maya S. Salnikova, C. Russell Middaugh, J. Howard Rytting
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS

Purpose To investigate protein-excipient interactions in lyophilized protein formulations. To evaluate the relationship between the extent of protein-excipient interactions and physico-chemical stability of protein.
Methods Growth Hormone was lyophilized with sucrose and trehalose in 1:2 weight ratio, and the formulations were stored at 50°C for 10 weeks. The protein-excipient interactions were analyzed immediately after lyophilization and during storage using Isoperibol Solution Calorimetry, Water Sorption Analysis and FTIR. The physical and chemical stability of the protein was monitored by RP-HPLC, SEC-HPLC and UV.
Results Protein formulations containing sucrose demonstrated higher protein-excipient interactions as compared to formulations containing trehalose. Although both formulations had similar chemical stabilities (deamidation) the physical stabilities (aggregation) were different. The sucrose-protein formulation had lower rates of aggregation.
Conclusion The high extent of the protein-excipient interactions is characteristic of homogeneous mixture of protein and excipient. It could be used as an additional parameter in evaluating the stability of protein formulations.

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41. Structural Studies of Phosphatase of Regenerating Liver (PRL-1) Using Multidimensional NMR Experiments
Andria Skinner, Jennifer Laurence
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS

PRL-1 was identified as an immediate early response gene in damaged liver tissue but has since been shown to be essential during development of many cellular tissues. This protein has also been found to promote cancer and trigger metastasis when over-expressed. PRL-1 activity is required for cell growth, but its cellular location differs between normal and cancerous cells, which commonly results from chemical modifications to a protein.
In the absence of reducing agents the conformation of PRL-1 favors a disulfide bond between C104 (the catalytic cysteine) and C49, which exhibits minimal enzymatic activity. For substrate binding to occur, the active site must open and expose the nucleophilic thiol. Addition of reducing agents exposes the catalytic motif, and specifically exposes Y53, which may be regulated by phosphorylation and impact phosphatase activity.
We investigated the role of Y53 and the disulfide bond in PRL-1 function by mutating C49 to serine to reduce the active site, Y53 to glutamic acid to mimic phosphorylation, and Y53 to phenylalanine to break the hydrogen bond with H103. NMR experiments made structural comparisons of the mutants to non-reduced and reduced wild type proteins. Kinetic experiments with p-nitrophenyl phosphate investigated PRL-1 enzymatic activity.
The NMR spectra of each protein have distinct differences. Enzymatically, the reduced and C49S proteins both exhibit increased activity compared to the non-reduced protein. Because the NMR spectra of C49S and the reduced wild type are different, reducing agents were added to C49S. Although the NMR spectra of the reduced C49S and non-reduced C49S are very similar, the enzymatic activity of C49S is inhibited with the addition of DTT. Y53E and Y53F have similar NMR spectra and exhibit greatly diminished activity compared to the wild type. The data indicate that PRL-1 is modulated by redox conditions at a second site, in addition to the disulfide bond between the nucleophilic C104 and partner C49, and phosphorylation at Y53 would inhibit phosphatase activity.

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42. Musashi and APC Cooperate to Maintain Colon Stem Cell Homeostasis
Lauren Baehner, Erick Spears, Kimberly Honeycutt, Shane Stecklein, Anna Wang, Kristi Neufeld
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

Colorectal cancer is the second leading cause of cancer related deaths in the United States. Approximately 80% of all colon cancers are associated with a mutation in the adenomatous polyposis coli (Apc) gene. These two observations have led us to investigate the intracellular role of normal APC and how truncation of APC, the most common type of carcinogenic mutation, leads to the formation of precancerous polyps in the intestines of mice and humans. The studies presented here are particularly concerned with the potential role of APC in stem cell homeostasis and its regulatory relationship with a known stem cell marker, Mushashi-1 (MSI-1). The Musashi family of sequence specific RNA binding proteins has been shown to be involved in neural stem cell maintenance in fruit flies and mammals. In the mammalian system, MSI-1 binds to the 3’ untranslated region (3’UTR) of the m-numb mRNA and inhibits its translation. This translational regulation of m-numb leads to symmetric division of the stem cell, whereas the presence of m-numb inhibits Notch signaling leading to an asymmetric division yielding one stem cell and one partially differentiated cell. In mice, MSI-1 has also been observed as an intestinal stem cell marker. Previous studies have shown that Apc knockout mice show greater than twelve-fold increase in msi-1 mRNA in intestinal crypts. Our preliminary results indicate a reciprocal regulatory relationship between MSI-1 and APC. Short-interfering RNA (siRNA) knockdown of msi-1 significantly increases APC protein production but does not impact Apc mRNA in cultured colon cells. Somewhat unexpectedly, siRNA knockdown of Apc in the same cells leads to an increase in both msi-1 transcript and protein product. These results, combined with others, have inspired the hypothesis that Musashi family members (MSI-1 in particular) cooperate with APC to regulate asymmetric division and differentiation of colorectal stem cells. Following this line of thinking, we would expect deregulation of this relationship by the loss of APC to play a role in colorectal carcinogenesis.

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43. Fluorous Phototriggers: Efficacy, Variability, and Mechanistic Features
Kenneth F. Stensrud, Richard S. Givens
Department of Chemistry, University of Kansas, Lawrence, KS

Photoremovable protecting groups (ppg) afford both temporal and spatial control over release of tethered substrates. In this process, light is employed to sever covalent linkages between a chromophore and substrate, thereby reinstating substrate functionality. Our group has demonstrated that the photochemical and photophysical properties of p-hydroxyphenacyl (pHP) are very favorably disposed toward rapid, efficient, and clean photorelease of a variety of bioactive substrates. For example, the photolysis of pHP-g-aminobutyric acid (pHP-GABA) occurs with greater than 20% efficiency for release with a rate const. of 1.82 x 108 s-1. Further studies have revealed improvements in these properties when electron withdrawing groups are attached to the pHP-GABA. In efforts to extend investigations of the electronic effects on the photorelease process, several new fluorinated pHP derivatives were synthesized and photolyzed. The efficiencies and rates have been detected. In addition to the photochemical studies, novel synthetic routes to these fluorinated chromophores will be highlighted.

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44. Shigella flexneri IpaBC Binds Preferentially to Membranes Containing Cholesterol
C. M. Terry, A. T. Harrington, N. Darboe, W. D. Picking
Department of Molecular Biosciences, University of Kansas, Lawrence, KS

Shigella flexneri is a bacterial pathogen that is the major cause of bacillary dysentery, a potentially life-threatening disease among children in the developing world. To promote bacterial entry into epithelial cells in the colon, the first step in shigellosis, S. flexneri secretes a number of protein effectors that are transferred to the host cell via a ‘type III secretion system’ (TTSS). Two of these effector proteins, the invasion plasmid antigens IpaB and IpaC, form a complex that becomes inserted into the host cell cytoplasmic membrane. Until now, the formation of a pore by IpaB and IpaC has only been demonstrated indirectly. Both proteins will interact with and destabilize liposomes in vitro and other groups have shown that IpaB binds preferentially to areas of the plasma membrane rich in cholesterol. However, the ability of IpaB-IpaC complexes purified from S. flexneri supernatants has not been well-characterized and their effect on liposomes remains unknown. The ability of a protein to bind and disrupt the integrity of mixed component liposomes can be measured through a novel dye-release assay and we use such an assay here to characterize the interaction of IpaC, IpaB and IpaB-IpaC complexes with liposomes composed of both acidic and neutral phospholipids. We also describe the mechanism of dye release caused by the IpaB-IpaC complex, which is reminiscent of the formation of a port that is consistent with that inserted into erythrocytes by S. flexneri. The importance of IpaB and IpaC in epithelial cell invasion by S. flexneri is described in both general mechanistic terms and in their temporal appearance at the pathogen-host interface.

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45. Nickel Influence on PRL-1 C-terminal Farnesylation: Implications for Localization
Natalie A. Ciaccio, Anthony A. Vartia
Department of Pharmaceutical Chemistry, University of Kansas, Lawrence, KS

Phosphatase of Regenerating Liver-1 (PRL-1) was originally identified as an early response gene promoting liver regeneration in rats after partial hepatectomy. Since then, overexpression of PRL-1 has been found to trigger cancer metastasis and promote tumor formation. While the biological function of PRL-1 remains largely unknown, data has shown the localization of PRL-1 to be highly variable. It has been found in the nucleus, endosome, and cytoplasm as well as at the mitotic spindle and plasma membrane. These changes appear to be cell-cycle dependent and tissue specific. Farnesylation at a C-terminal –CaaX motif and electrostatic interactions of a polybasic region are thought to control PRL-1’s association with the plasma membrane. UV-Vis Absorption spectra and inductively-coupled plasma mass spectrometry (ICP-MS) analysis of PRL-1 –CaaX mutants indicate that cysteines in this motif also bind nickel when recombinantly expressed in E. coli and purified by nickel-iminiodiacetic acid (Ni-IDA) resin. Additionally, recombinant expression of PRL-1 in S. cerevisiae, which are biochemically capable of performing isoprenylation and downstream processing, results in localization of PRL-1 at the plasma membrane. We aim to confirm the farnesylation status of PRL-1 recombinantly expressed in S. cerevisiae and determine differential localization with and without the farnesyl moiety. We further hope to study whether or not the presence of Ni or other metals present in the cell affects the farnesylation of PRL-1 and its subsequent localization and function.

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University of Kansas
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