Master of Science / Biochemistry / John Tomich / One of the important keys to characterize the biological function of a protein is the study of post-translational modification (PTM). Formation of disulfide bond linkages between cysteine residues within a protein is a common PTM which not only contributes to folding and stabilizing the protein structure, but also to accomplishing its native function. Therefore, the study and discovery of structural-functional relationships of expressed proteins using an isolated proteomics approach has been one of the biggest advances within the field of structural biology in recent years. In this study, rapid disulfide bond mapping of freshly obtained equine serum albumin (ESA) was performed using matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). Highly sensitive MALDI-TOF MS is commonly used for the investigation of disulfide bond linkages in the proteomics field. However, it has also been known that the presence of disulfide bond linkages absorbs the energy which is created by the cysteine-cysteine kinetic vibration, resulting in a decrease of the instrumental sensitivity. To overcome this problem, the disulfide bond mapping method was optimized by applying a combination of chemical labeling, proteolytic enzymes, and matrices. With the optimized method, we were also able to achieve high protein sequence coverage. Obtaining higher sequence coverage of a protein provides more information about a protein which helps to identify the protein by peptide mass fingerprint (PMF) technique. These analyses eventually contribute to the estimation of the possible PTM sites.

Doctor of Philosophy / Department of Chemistry / Paul E. Smith / In the study of biological systems, molecular dynamics (MD) simulations have played an important role in providing atomic details for phenomena of interest. The force field used in MD simulations is a critical factor determining the quality of the simulations. Recently, Kirkwood-Buff (KB) theory has been applied to study preferential interactions and to develop a new force field. KB theory provides a path from quantities determined from simulation data to the corresponding thermodynamic data. Here we combine KB theory and molecular simulations to study a variety of intermolecular interactions in solution. First, recent results concerning the formulation and evaluation of preferential interactions in biological systems in terms of KB integrals are presented. In particular, experimental and simulated preferential interactions of a cosolvent with a biomolecule in the presence of water are described. Second, a force field for the computer simulation of aqueous solutions of amides is presented. The force field is designed to reproduce the experimentally observed density and KB integrals for N-methylacetamide (NMA), allowing for an accurate description of the NMA activity. Other properties such as the translational diffusion constant and heat of mixing are also well reproduced. The force field is then extended to include N,N'-dimethylacetamide and acetamide with good success. The models presented here provide a basis for an accurate force field for peptides and proteins. Comparison between the developed KB force fields (KBFF) and existing force fields is performed for amide and glycine and proves that the KBFF approach is competitive. Also, explicit expressions are developed for the chemical potential derivatives, partial molar volumes, and isothermal compressibility of solution mixtures involving four components at finite concentrations using the KB theory of solutions. A general recursion relationship is also provided which can be used to generate the chemical potential derivatives for higher component solutions. Finally, a pairwise preferential interaction model (PPIM), described by KB integrals is developed to quantify and characterize the interactions between functional groups observed in peptides.

Master of Science / Department of Biochemistry / John M. Tomich / Biologically based adhesives offer many industrial advantages over their chemically synthesized counterparts, not the least of which are reduced environmental impact and limited toxicity. They also represent a renewable resource. In addition, nanoscale biomaterials also show an incredibly large potential for biomedical uses, including possible drug delivery and novel wound bandaging, as well as tissue engineering. Understanding the adhesion mechanisms at work in peptide-based nanomaterials is key for producing viable industrial and clinical biomimetic compounds. Our previous work has shown that small hydrophobic oligopeptide segments flanked by short tri-lysine sequences display adhesion strength that is dependent on the formation of β-structure and large-scale association of monomers. In this study, three oligopeptides were synthesized based on putative amyloid fibril nucleation sites. Two of the sequences originate from the Alzheimer’s beta amyloid peptide Aβ1-40, while the third sequence comes from a nucleation site for islet amyloid polypeptide (IAPP). These peptides show unusual structural properties associated with adhesive ability. Furthermore, they represent a third category of requirements for β-structure formation. In addition, I report the first morphological evidence for the previously predicted structural mechanism underlying our previous peptide based adhesives.

Doctor of Philosophy / Department of Biochemistry / Anna Zolkiewska / Muscle satellite cells are the primary stem cells of postnatal skeletal muscle. Quiescent satellite cells become activated and proliferate during muscle regeneration after injury. They have the ability to adopt two divergent fates: differentiation or self-renewal. The Notch pathway is a critical regulator of satellite cell activation and differentiation. Notch signaling is activated upon the interaction of a Notch ligand present in a signal-sending cell with a Notch receptor present in a signal-receiving cell. Delta-like 1 (Dll1) is a mammalian ligand for Notch receptors. In this study, we found that Notch activity is essential for maintaining the expression of Pax7, a transcription factor associated with self-renewing satellite cells. We also demonstrated that Dll1 represents a substrate for several ADAM metalloproteases. Dll1 shedding takes place in a pool of Pax7-positive self-renewing cells, but Dll1 remains intact in differentiated myotubes. Inhibition of Dll1 shedding with a dominant-negative form of ADAM12 leads to elevated Notch signaling, inhibition of differentiation and expansion of the pool of self-renewing cells. We propose that ADAM-mediated shedding of Dll1 helps achieve an asymmetry in Notch signaling in initially equivalent myogenic cells and helps sustain the balance between differentiation and self-renewal. Pax7 plays a key role in protecting satellite cells from apoptosis. The mechanism of Pax7 protecting muscle satellite cells from apoptosis is not well understood. In the second part of this study, we show that Pax7 up-regulates manganese superoxide dismutase (MnSOD) at the transcriptional level, suggesting the involvement of MnSOD in Pax7-mediated cell survival. A specific chromosomal translocation involving the Pax7 gene and generation of a fusion protein Pax7-FKHR is found a childhood cancer, rhabdomyosarcoma. Furthermore, the level of the wild-type Pax7 is down-regulated in rhabdomyosarcomas. In the third part of this dissertation, we investigated the dominant-negative effect of Pax7-FKHR fusion protein on the wild-type Pax7, and found that the Pax7 protein level is down-regulated by Pax7-FKHR expression while the Pax7 mRNA level is not affected. We propose a specific microRNA-mediated inhibition of Pax7 mRNA translation by the oncogenic Pax7-FKHR fusion protein.

Doctor of Philosophy / Department of Chemistry / Paul E. Smith / In the past several decades, molecular dynamics (MD) simulations have played an important role in providing atomic details for phenomena of interest. The force field used in MD simulations is a critical factor determining the quality of the simulations. Kirkwood-Buff (KB) theory has been applied to study preferential interactions and to develop a new force field. KB theory provides a path from quantities determined from simulation data to the corresponding thermodynamic data. Here we combine KB theory and molecular simulations to study a variety of intermolecular interactions in solution. First, a force field for the computer simulation of aqueous solutions of alcohols is presented. The force field is designed to reproduce the experimentally observed density and KB integrals for a series of alcohols, allowing for an accurate description of alcohols’ activity. Other properties such as the translational diffusion constant and heat of mixing are also well reproduced. Second, the newly developed force field is then extended to more complicated systems, such as peptide or mini-proteins, to determine backbone dihedral potentials energetics. The models developed here provide a basis for an accurate force field for peptides and proteins. Third, we have then studied the surface tension of a variety water models. Results showed that different simulation conditions can affect the final values of surface tension. Finally, by using the Kirkwood-Buff theory of solution and surface probability distributions, we attempted to characterize the properties of the Gas/Liquid interface region. The same approach is then used to understand the relationship between changes in surface tension, the degree of surface adsorption or depletion, and the bulk solution properties.

Doctor of Philosophy / Department of Biochemistry / Lawrence C. Davis / The fate and bioavailability of organic contaminants in plants is a major ecological and human health concern. Current wet chemistry techniques that employ strong chemical treatments and extractions with volatile solvents, such as GC-MS, HPLC, and radiolabeling, although helpful, degrade plant tissue resulting in the loss of spatial distribution and the production of artifacts. Synchrotron radiation infrared microspectroscopy (SR-IMS) permits direct analysis of plant cell wall architecture at the cellular level in situ, combining spatially localized information and chemical information from the IR absorbances to produce a chemical map that can be linked to a particular morphology or functional group. This study demonstrated the use of SR-IMS to probe biopolymers such as cellulose, lignin, and proteins in the root tissue of hydroponically grown sunflower and maize plants as well as to determine the fate and effect of several organic contaminants in those root tissues. Principal components analysis (PCA), a data compression technique, was employed to reveal the major spectral variances between untreated and organic contaminant treated root tissues. Treatment with 1H-benzotriazole (BT) caused alterations to the lignin component in the root tissue of plants. The BT was found in xylem and epidermal tissue of sunflower plants but not associated with any particular tissue in maize roots. 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) altered the pectin and polysaccharide structure in both maize and sunflower. SR-IMS revealed the reduction of DNTs to their aromatic amine form in the vascular and epidermal tissues at low concentration. At high concentration, DNTs appeared to be associated with all the plant tissues in maize and sunflower. Exposure of sunflower and maize to 2,6-dichlorophenol (2,6-DCP) caused alterations to the polysaccharide and protein component of the root tissue. In some cases, phenolic compounds were observed in the epidermal tissue of maize and sunflower roots. The results of this research indicate that SR-IMS has the potential to become an important analytical tool for determining the fate and effect of organic contaminants in plants.

Doctor of Philosophy / Department of Biochemistry / Karl J. Kramer / Subbarat Muthukrishnan / Chitin, a linear homopolymer of beta-1,4-linked N-acetylglucosamine, is the second most abundant biopolymer next to cellulose. It is the major structural polysaccharide in the insect’s exoskeleton and gut lining. An extensive study of two of the major genes encoding enzymes involved in chitin metabolism, chitin synthases (CHSs) and beta-N-acetylglucosaminidases (NAGs), was undertaken. CHS genes from the tobacco hornworm, Manduca sexta, and NAG genes from the red flour beetle, Tribolium castaneum, were identified and characterized. In general, chitin deposition occurs in two major extracellular structures of insects, the cuticle that overlays the epidermis, and the peritrophic membrane (PM) that lines the midgut. Only two CHS genes were identified in M. sexta using Southern blot analysis. Extensive expression studies of both M. sexta CHS genes, MsCHS1 and MsCHS2, suggest a strict functional specialization of these two genes for the synthesis of epidermal and PM-associated chitin, respectively. Furthermore, two alternatively spliced transcripts of MsCHS1, MsCHS1a and MsCHS1b, were identified. Analysis of the levels of these transcripts in different tissues and stages of development indicated that the MsCHS1a transcript predominates in the integument during the feeding and pupal stages, whereas the MsCHS1b transcript is more abundantly present in the tracheae, foregut, and hindgut during all developmental stages tested. Four genes encoding putative NAGs (TcNAG1, TcNAG2, TcNAG3, and TcNAG4) were identified by searching the Tribolium genomic database. The full-length cDNAs for all four NAGs were cloned and sequenced, and the exon-intron organizations were determined. Studies on developmental expression patterns of each gene indicated that they are expressed during most developmental stages with TcNAG1 being the predominant one. The function of each NAG was assessed by down regulating the level of each transcript at various developmental stages using RNA interference. Selective knock down of each transcript, without significant reduction in the expression levels of the other NAG transcripts, was verified and the resulting phenotypes were documented. Knockdown of TcNAG1 interrupted larval-larval, larval-pupal, and pupal-adult molting, and the insects were unable to completely shed their old cuticles.

Doctor of Philosophy / Department of Entomology / Michael R. Kanost / James R. Nechols / Cellular encapsulation is the immune response in which insects protect themselves from multicellular parasites such as nematodes or parasitoids. During an encapsulation episode, certain insect hemocytes become attracted to a foreign invader and aggregate on its surface. In short order, the invading entity will become entrapped within a capsule comprised of thousands of hemocytes, thus rendering the parasite harmless to the insect host. Although the process of cellular encapsulation has been known for a great many years, very little knowledge yet exists regarding the biochemistry underlying capsule formation. It would seem likely that cell surface adhesion proteins mediate this immune response. In a series of in vivo encapsulation assays in the tobacco hornworm, Manduca sexta, a collection of anti-hemocyte monoclonal antibodies (mAbs) was screened for their ability to inhibit cellular encapsulation. Two of the mAbs that inhibited this immune response and incidentally specifically bind plasmatocytes, MS13 and MS34, were used to isolate a ≈ 90 kDa protein. Several short peptide sequences contained within this protein were acquired via Edman degradation. Degenerate primers based on two of these peptide sequences and total RNA from M. sexta hemocytes were used to perform RT-PCR and 5´ and 3´ RACE. This resulted in a full-length cDNA sequence of 2426 bp. A 2301 bp open reading frame within this cDNA sequence codes for a protein of 767 residues. This protein, denominated [Beta]Ms1, exhibits significant sequence homology to the [Beta]-subunits of integrins, which are a family of transmembrane, heterodimeric glycoproteins that possess adhesive properties. Analysis of recombinant segments of [Beta]Ms1 showed that the protein produced from the PCR product is the antigen to MS13 and MS34 and that these mAbs bind to the region of the integrin that contains the extracellular binding site. Northern blot analysis of various M. sexta tissues together with immunofluorescence labeling with MS13 and MS34 shows that [Beta]Ms1 is solely expressed in plasmatocytes. The totality of these experiments demonstrates that integrins are essential for the cellular immune response of encapsulation.

Doctor of Philosophy / Biochemistry Interdepartmental Program / Qize Wei / This dissertation describes the role of myosin-interacting guanine nucleotide exchange factor (MyoGEF) and centrosome/spindle pole associated protein (CSPP) in mitotic progression and cytokinesis. We have identified three mouse isoforms of CSPP, all of which interact and colocalize with MyoGEF to the central spindle in anaphase cells. The N-terminus of MyoGEF interacts with myosin whereas the C terminus interacts with the N-terminus of CSPP, forming a complex. The N-terminus of CSPP appears to be important for both localization and interaction with MyoGEF. CSPP plays a role in mitotic progression since its depletion by RNAi resulted in metaphase arrest. MyoGEF is required for completion of cytokinesis. Both MyoGEF and CSPP are phosphorylated by mitotic kinases including Plk1 and Aurora. Importantly, MyoGEF is phosphorylated at Thr-574 in mitosis by Polo-like kinase 1, and this phosphorylation is required for activation of RhoA. Thr-543 of MyoGEF is required for Plk1 binding in mitosis and phosphorylation of MyoGEF by Cdk1/cyclinB, possibly at Thr-543 may generate a Plk1 docking site, i.e., Cdk1 can phosphorylate MyoGEF at Thr-543, thereby allowing Plk1 to bind and phosphorylate MyoGEF at Thr-574. Finally, MyoGEF and CSPP are also phosphorylated by Aurora-B kinase in vitro. Taken together, we propose that Aurora-B may phosphorylate and recruit MyoGEF and CSPP to the central spindle, where phosphorylation of MyoGEF at Thr-543 promotes Polo kinase binding and additional phosphorylation of MyoGEF, leading to the activation of RhoA at the cleavage furrow.

Doctor of Philosophy / Department of Biochemistry / Michael R. Kanost / Manduca sexta is a large insect species well-suited for biochemical analysis of proteins in the hemolymph (blood) that respond to infection. Insects lack adaptive immunity and rely entirely on innate immunity to prevent and manage infection. Immune response proteins include proteins that bind pathogens and activate serine proteases, which function in proteolytic cascades that trigger effector responses, such as antimicrobial peptide production and prophenoloxidase activation. Phenoloxidase catalyzes melanin synthesis, which leads to microbial killing. I used MALDI-TOF/TOF mass spectrometry and immunoblotting to identify M. sexta proteins present in putative immune complexes. From analyses of high molecular weight gel filtration fractions of plasma activated by microbial polysaccharides, I detected hemocytin, prophenoloxidase, and cleaved serine protease homologs, suggesting prophenoloxidase and serine protease homologs form large complexes in plasma. I used in vitro bacterial binding assays to identify hemolymph proteins that bind either directly or indirectly to the surface of bacteria or curdlan. Prophenoloxidase, annexin IX, and hemocyte aggregation inhibitor protein were found bound to all the samples tested, indicating they play a role in the early stage of immune response. Serpins regulate specific active proteases by covalently binding and forming serpin-protease complexes. Serpin-1, an abundant plasma protein, has an alternatively spliced ninth exon encoding 12 serpin-1 isoforms that differ in inhibitory selectivity. RT-PCR showed that all 12 isoforms are expressed in hemocytes, fat body, and midgut. Comparisons of naïve and immune-challenged hemocytes and fat body indicated the immune-related upregulation of serpin-1A but not the other isoforms. Using immunoaffinity chromatography I isolated two serpin-1-protease complexes from plasma after activation with bacterial lipopolysaccharide. MALDI-TOF/TOF analysis of these serpin-1-protease complexes identified the digestive enzyme chymotrypsin as a specific target of serpin-1K. Nine out of the twelve serpin-1 isoforms were identified from control plasma at the protein level using 2D-PAGE. Serpin-1 protease complexes were identified by 2D-PAGE analysis: serpin-1A, E and J were found to be complexed with hemolymph proteinase-8 and an unidentified isoform of serpin-1 was complexed with hemolymph proteinase-1. Discovering the serpin-1 isoforms that inhibit specific proteases enhances our understanding of the regulation of proteolytic cascades in M. sexta.