Purified Schwann cells were cultured from neonatal rat sciatic nerve using a modification of the method of Brockes (Brockes, J.P. et al, Brain Res. 165(1979) 105-118). Schwann cells and contaminating fibroblasts were unambiguously identified using fluorescent antibodies to 2'3' cyclic nucleotide 3'-phosphodiesterase and the thy 1.1 antigen respectively. The Schwann cells were quiescent unless challenged with mitogens, They proliferated rapidly in response to the soluable mitogen, cholera toxin, or to membrane fractions from rat CNS or PNS, prepared by the method of DeVries (DeVries, G.H., J Neurochem, 40 (1983) 1709-1717). Mitogenic activity was present in both axolemmal and myelin enriched fractions and promoted a 10-15 fold increase in the rate of 3H-thymidine uptake. The axolemmal mitogen was sensitive to heat (800C for 10 minutes), trypsin digestion (0.05% x 30 mins) or to treatment with endoglycosidase D, suggesting that it could be a glycoprotein. Fifty percent of the axolemmal mitogenic activity was solubilized in 1% octyl-glucoside. The solubilized material, however, was very unstable and further purification was not possible. The myelin associated mitogenic activity was markedly different. It was resistant to freeze thaw cycles, trypsin digestion or endoglycosidase treatment and the activity was actually enhanced by heating at 100°C for two hours. It is proposed that the axolemmal activity is responsible for Schwann cell proliferation during development and that the myelin associated activity promotes Schwann cell proliferation during Wallerian degeneration.

Phosphatidylserine (PS) is exposed on a variety of tumour cell types, virus-infected cells and virions. Bavituximab, a PS-targeting antibody with a human immunoglobulin G (IgG) constant region, possesses anti-viral and anti-cancer activity that is mainly mediated by antibody-dependent cell-mediated cytotoxicity (ADCC). ADCC of therapeutic antibodies is naturally limited in vivo by the prevailing serum concentrations of IgG which compete for Fcγ receptor (FcγR) binding. The binding of bavituximab to FcγRIIIA is shown here to be reduced in the presence of either whole serum or polyclonal IgG. However, the use of IdeS, a highly specific IgG-cleaving enzyme derived from Streptococcus pyogenes, presents a potential solution to this problem. IdeS cleaves in the hinge region of IgG between amino acid residues Gly236 and Gly237 but is thought to possess an additional binding interface with its IgG substrate. Firstly, the location of the IdeS–IgG binding interfaces was investigated using truncated IgG1 variants and systematic amino acid mutagenesis. IdeS bound an IgG1 fragment crystallisable (Fc) domain lacking the hinge region, thereby confirming the presence of a secondary binding site. Secondly, residues involved in the IdeS–IgG interaction were mutated to create an IdeS-resistant form of bavituximab. Pre-treatment of competing IgG with IdeS fully restored binding of IdeS-resistant bavituximab to FcγRIIIA, while pre-treatment of serum produced a partial improvement in binding. Lastly, bavituximab, the PS receptor TIM-1, and the related protein TIM-4 were tested for binding to a model alphavirus, Semliki Forest Virus. Only TIM-1, the smallest of these constructs, was found to bind the virus; this implies that receptor size may be an important consideration in the design of PS-targeting anti-viral agents. Overall, these data suggest possible methods for improving the therapeutic targeting of PS, either by optimisation of antigen binding regions or by enhancement of FcγR binding.

<p>The MazEF toxin-antitoxin (TA) system consists of the antitoxin MazE and the toxin MazF. MazF is a sequence-specific endoribonuclease that upon activation causes cellular growth arrest and increass the level of persisters. Moreover, MazF-induced cells are in a quasi-dormant state that cells remain metabolically active while stop dividing. The quasi-dormancy is similar to the nonreplicating state of M. tuberculosis during latent tuberculosis, thus suggesting the role of mazEF in M. tuberculosis dormancy and persistence. M. tuberculosis has nine mazEF TA modules, each with different RNA cleavage specificities and implicated in selective gene expression during stress conditions. To date only the Bacillus subtilis MazF-RNA complex structure has been determined. As M. tuberculosis MazF homologues recognize distinct RNA sequences, their molecular mechanisms of substrate specificity remain unclear. By taking advantage of X-ray crystallography, we have determined structures of two M. tuberculosis MazF-RNA complexes, MazF-mt1 (Rv2801c) and MazF-mt3 (Rv1991c) in complex with an uncleavable RNA substrate. These structures have provided the molecular basis of sequence-specific RNA recognition and cleavage by MazF toxins.</p><p>Both MazF-mt1-RNA and MazF-mt3-RNA complexes showed similar structural organization with one molecule of RNA bound to a MazF-mt1 or MazF-mt3 dimer and occupying the same pocket within the MazF dimer interface. Similar to B. subtilis MazF-RNA complex, MazF-mt1 and MazF-mt3 displayed a conserved active site architecture, where two highly conserved residues, Arg and Thr, form hydrogen bonds with the scissile phosphate group in the cleavage site of the bound RNA. The MazF-mt1-RNA complex also showed specific interactions with its three-base RNA recognition element. Compared with the B. subtilis MazF-RNA complex, our structures showed that residues involved in sequence-specific recognition of target RNA vary between the MazF homologues, therefore explaining the molecular basis for their different RNA recognition sequences. In addition, local conformational changes of the loops in the RNA binding site of MazF-mt1 appear to play a role in MazF targeting different RNA lengths and sequences. In contrast, the MazF-mt3-RNA complex is in a non-optimal RNA binding state with a symmetry-related MazF-mt3 molecule found to make interactions with the bound RNA in the crystal. The crystal-packing interactions were further examined by isothermal titration calorimetry (ITC) studies on selected MazF-mt3 mutants. Our attempts to utilize a MazF-mt3 mutant bearing mutations involved in crystal contacts all crystallized with few nucleotides, which are still found to interact with a symmetry mate. However, these different crystal forms revealed the conformational flexibility of loops in the RNA binding interface of MazF-mt3, suggesting their role in RNA binding and recognition, which will require further studies on additional MazF-mt3-RNA complex interactions.</p><p>In conclusion, the structures of the MazF-mt1-RNA and MazF-mt3-RNA complexes provide the first structural information on any M. tuberculosis MazF homologues. Supplemented with structure-guided mutational studies on MazF toxicity in vivo, this study has addressed the structural basis of different RNA cleavage specificities among MazF homologues. Our work will guide future studies on the function of other M. tuberculosis MazF and MazE-MazF homologues, and will help delineate their physiological roles in M. tuberculosis stress responses and pathogenesis.</p> / Dissertation

<p>Transcription factors (TF) interact with DNA to regulate gene expression by specifically binding one or two sequences out of millions of possible sites in a genome. Sequence specific binding (recognition) arises from two mechanisms: amino acid-nucleobase H-bonding (direct readout), and sequence-dependent DNA deformability and structure (indirect readout). These lead to tight, specific binding. TFs can also rapidly search for their target by attaching to any sequence and then sliding along DNA. This decreases the space through which a TF randomly walks to stumble on the target. Tight binding and rapid sliding are optimized because TFs can switch between different DNA-binding modes, a search mode and a recognition mode. Due to the disorder during search and the dynamics during recognition, the atomic level dynamics are too transient to see by experimental techniques. Therefore my goal was to develop an atomistic model of protein-DNA search and recognition using computer simulations. To meet this goal I studied a model TF, the human mitochondrial transcription termination factor-1 (MTERF1), which has a modular superhelical-architecture complementary to DNA. I used molecular dynamics (MD) simulations to characterize MTERF1 search and recognition, alongside other computational methods to generate my structural models. I was the first to observe spontaneous sliding of a TF on DNA. I found that flexibility between modules permitted contacts along the DNA footprint to shift independently. This implies that the net sliding barrier of MTERF1 is similar to the barrier for one module-DNA contact rather than the sum of all module-DNA contacts. Next I utilized enhanced sampling to drive MTERF1 over the recognition barrier(s). I found that the DNA deformed as MTERF1 made increasingly more direct readout contacts to DNA, with the global recognition process characterized by partnered DNA and MTERF1 helix unwinding. These results suggest that direct and indirect readout occur simultaneously during recognition as MTERF1 unwinds (deforms) the target DNA sequence. Helix unwinding motions were present in search mode, suggesting the dynamics that drive search and recognition are intrinsic to the MTERF1 architecture.

No description available.

BIOCHEMISTRY Oncogenic KRAS and BRAF Drive Metabolic Reprogramming in Colorectal Cancer Josiah Ewing Hutton, III. Dissertation under the direction of Professor Daniel C. Liebler Analysis of cancer cells that have undergone metabolic reprogramming, where glucose metabolism is altered to support rapid proliferation, is typically performed utilizing transcriptomic and metabolomic techniques. Transcriptomic data does not always accurately reflect protein expression levels, and metabolomic data alone does not sufficiently explain how these cancer cells have undergone metabolic reprogramming. Mutations in the oncogenes KRAS and BRAF induce metabolic reprogramming in colorectal cancers through enhanced glucose transport, but the broader impact of these oncogenes on metabolic pathways is unclear. I hypothesized that mutant KRAS and mutant BRAF drive metabolic reprogramming in colorectal cancer cells by diverting glucose and glutamine carbons to the production of biosynthetic precursors for rapid growth and that this metabolic reprogramming can be detected by mass spectrometry based proteomic methods. These methods were used, in conjunction with RNA-Seq and metabolite measurements, to determine if isogenic DLD-1 and isogenic RKO cell lines had undergone metabolic reprogramming driven by mutant KRAS G13D and mutant BRAF V600E, respectively. Glucose consumption and lactate production rates indicated that the isogenic cells expressing the mutant oncogenes had undergone metabolic reprogramming. The global transcriptomic and shotgun proteomic analyses did not reveal any metabolic reprogramming at the mRNA or protein levels. However, more sensitive, precise, targeted proteomic analysis of 73 metabolism proteins in these cell lines revealed biologically important protein expression fold changes in proteins involved in glucose metabolism, glutamine metabolism, and the serine biosynthesis pathways. A study of 8 KRAS wild type and 8 KRAS mutant human colon tumors confirmed the association of increased expression of glycolytic and glutamine metabolic proteins with KRAS mutations. These results demonstrate that mutant KRAS and mutant BRAF drive metabolic reprogramming through modest protein expression changes. Moreover, targeted proteomics was required to measure small protein expression differences that could not be detected by transcriptomics or shotgun proteomics. Measurement of the mutant and wild type KRAS and BRAF proteins indicated that the abundance of wild type and mutant BRAF proteins reflected the allelic composition of the RKO cells, whereas wild type and mutant KRAS protein levels did not in DLD-1 cells. Levels of mutant KRAS G13D protein did not reflect the degree of metabolic reprogramming in the DLD-1 cell lines, whereas levels of mutant BRAF V600E protein were proportional to the degree of metabolic reprogramming in the RKO cells. These results underscore the importance of performing targeted, quantitative protein measurements to evaluate the impact of oncogenic mutations. Approved Date Daniel C. Liebler, Ph.D.

<p> A distinguishing feature of enzymatic catalysis in comparison to small-molecule catalysts is that enzymes use non-covalent interactions to position substrates and active site residues relative to each other. Although residues implicated in positioning can be readily identified by structural inspection, understanding the catalytic importance of these interactions requires experimental tests. Enzyme active sites often contain networks of interactions for which the functional role cannot be distinguished by the structure alone. In triosephosphate isomerase (TIM), a key enzyme in glycolysis, structural and mutational results suggest an important catalytic role for three active site residues: Lys13, His95, and Glu167. A glutamate residue (Glu97) has been shown to be conserved in almost all species of TIM. Based on X-ray structures, a potential role of Glu97 may be to position Lys13. To investigate the role of Glu97 in TIM catalysis, we mutated Glu97 to Ala, Gln, and Asp in <i> Trypanosoma brucei brucei</i>. The Glu97Ala, Glu97Gln, and Glu97Asp mutations led to an ~10,000-fold, ~40-fold, and ~10-fold reduction in <i>k_cat</i> respectively. The similar K_M value in Glu97Gln and Glu97Asp mutations relative to wild-type TIM suggests substrate binding is not affected by the mutation. The Glu97Ala mutation led to a slight increased <i> K_M</i> relative to wild-type, suggesting that Glu97 may play a role in maintaining the structural stability of TIM. Circular dichroism analysis shows that the E97 mutations do not affect the overall helical structure of TIM. Overall, the results provide evidence for this non-active site residue playing an important role in TIM catalysis.</p>

Oxidative stress is a contributing factor in a number of chronic diseases, including cancer, atherosclerosis, and neurodegenerative diseases. Lipid peroxidation that occurs during periods of oxidative stress and decomposition of these oxidized lipids results in the formation of lipid electrophiles. 4-Hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) are two lipid aldehydes which are generated as a result of lipid peroxidation, and both can adduct nucleophilic side chains of amino acids in proteins. A large number of protein targets have been identified for HNE and ONE, consisting of an array of adduct structures Here, we show that these adducts have distinct functional implications on the activity and regulation of the target protein. CDK2, a key cell cycle kinase which regulates the G1/S-phase transition, is adducted by HNE for up to 16 h. The adduction of CDK2 inhibits kinase activity in vitro and in cells and delays cell cycle progression into S-phase following HNE treatment. PIN1 is a cis/trans isomerase, which plays a key role in regulating of a number of cell signaling pathways. PIN1 is modified by 4-oxo-2-nonenal ONE at the active-site Cys and forms a cross-link with a nearby Lys, thereby inactivating the protein. Using site-specific incorporation of deuterium in ONE, we were able to determine a mechanism of cross-link formation and definitively show that nucleophilic attack occurs at the third carbon of ONE. Histone proteins have also been shown to be preferential targets for ONE modification, and these proteins have a direct effect on the regulation of gene expression and chromatin structure. We have developed a method to selectively isolate regions of DNA associated with these adducted histones using click-chemistry. The method, coupled with next generation DNA sequencing, termed Click-Seq, shows few regions of enrichment, suggesting that ONE broadly adducts chromatin. Furthermore, the levels of these adducts are two orders of magnitude lower than the canonical histone modifications. Together, these data show that the lipid electrophile HNE and ONE can have a significant impact on enzyme activity, alterations in cell signaling pathway, and regulation of gene expression.

Osteoporosis is a disease characterized by low bone mineral density and overall weakening of bone. It shows sex association, affecting post-menopausal women at higher rates than men. It is also is age specific, showing increased prevalence with aging in both sexes. Due to the increased mortality and severe disruptions in quality of life caused by osteoporosis-related fractures, the disease poses a serious public health concern (Sözen, Özışık, & Başaran, 2017). While osteoporosis is known to be related to environmental factors and associated lifestyle factors, recent genome-wide association studies (GWAS) have found dozens of loci associated with low bone mineral density and increased fracture risk, necessitating further study into specific causal genes (Sabik & Farber, 2017; Zheng, Spector, & Richards, 2011). Bone self-renewal, throughout life, is facilitated by resident mesenchymal stem cell (MSC) populations found within bone tissues, which are capable of osteogenic differentiation. Establishment of reliable cell culture methods for the growth and differentiation of bone marrow MSCs is a pre-requisite to carrying out comparative population-level studies of MSCs focused on assessing the interactions between underlying genetic and co-morbidity features that affect their osteogenic potential. Culture media is often supplemented with fetal bovine serum (FBS), containing essential components that promote cell growth. However, use of FBS has led to issues in controllability and reproducibility of data from cultures supplemented with FBS because of its poorly-defined nature and variations in composition from sources of FBS. In this study, the efficacy of bone marrow-derived MSC (BMSC) cultures grown in a commercially available artificial media was compared to cells grown in media containing FBS, and their growth and osteogenic differentiation capacity were compared. Human MSCs were obtained from the acetabular reaming samples from 10 patients undergoing total hip arthroplasty at Boston Medical Center. We processed the raw materials to remove tissue and adipose materials, and seeded using the loose cellular component of the bone marrow in media supplemented with FBS and commercially available serum-free media, Mesencult-ACF (Stem Cell Technologies, Vancouver, Canada). Adherent cell MSC populations were selected over a seven-day growth period. Osteogenic differentiation was initiated using supplementation with Dexamethasone, ascorbate, and -glycerol phosphate. Cultured cells were grown in both media conditions for 21 days after osteogenesis was initiated. We examined cellular DNA content for cell growth, alkaline phosphatase (ALP) for osteogenic differentiation and calcium content for mineralization and terminal osteocyte differentiation. We did not find significant differences between the groups for average DNA, ALP or calcium content (p=0.167, p=0.139, p=0.291). We did find significant differences within each subject between groups for nearly every subject and across all measurements. We did find significant differences in the DNA content and ALP content of the FBS supplemented media group between subjects (p=0.045, p=0.020). Findings suggest Mesencult-ACF is a suitable alternative to media supplemented with FBS for promoting growth and differentiation of BMSCs.

Parkinson’s Disease is a neurodegenerative illness of the central nervous system that impacts both cognitive and motor functioning. Current understanding dictates that these symptoms are caused by the death of dopaminergic neurons within the substantia nigra pars compacta of the basal ganglia. This neuronal death explains part of the symptoms, but large clumps of insoluble protein called Lewy Bodies are thought to also contribute. The α-synuclein (α-SN) protein is a major component of Lewy Bodies and has long been demonstrated to misfold and become aggregated. Classically, these Lewy Bodies first appear within the brainstem and spread upwards towards the cortex as the pathology progresses. While there have been some suggestions, the mechanism whereby α-SN misfolds in the brainstem is not currently understood. Additionally, the interplay between Lewy Bodies and dopaminergic neuronal death has not yet been discovered. One of the leading theories of α-SN misfolding is related to the gut-brain axis. Our gastrointestinal tract is innervated by the enteric nervous system, which sends signals to the brainstem via the vagus nerve. Curiously, α-SN is expressed within enteroendocrine cells of the intestine. These cells have connections to nerve fibers of the enteric nervous system at their basal lamina. The hypothesis reasons that some pathogenic agent can gain access to the cells of the intestine, cause α-SN to misfold, and cause this damaged protein to be transported within the vagus nerve. As the vagus nerve ultimately connects to the brainstem, this is where the Lewy Bodies are deposited to initiate Parkinson’s disease. Research has investigated this connection thoroughly but has yet to determine definitively if this gut-brain axis is truly causing pathology within the brainstem. This thesis attempts to provide an overview of the current literature relating to Parkinson’s disease as well as the current knowledge of the gut-brain axis. Then, this will evaluate the various entities proposed to be the unknown pathogen of the gut-brain axis and discuss some of the controversies in the literature. Finally, this thesis will discuss the therapies that have arisen from the concept of the gut-brain axis and the potential further directions that these therapies imply for research.