Progesterone regulates female reproduction in many ways, yet it is still unclear how signals are conveyed through nuclear and extranuclear receptors. The traditional notion was that progesterone binds classical progesterone receptors to alter gene transcription. This view has been challenged by the discovery of additional progesterone signaling molecules important for progesterone actions in non-neural cells. In granulosa cells, the progesterone receptor membrane component 1 (Pgrmc1) mediates progesterone effects by forming a receptor complex with binding partner, Serpine mRNA binding protein 1, but it is unknown whether these molecules function similarly in the brain. To begin to address these issues, I investigated the neural role of Pgrmc1 in female mouse brain, rat brain and in neural cells. By examining the neuroanatomical localization, hormonal regulation, and colocalization of Pgrmc1 within key neurons in the neural control of ovulation, Pgrmc1 emerged as a candidate signaling molecule likely to mediate progesterone functions. Furthermore, Pgrmc1 levels regulate the expression of several diverse genes and signaling pathways in neural cells. Taken together, these results demonstrate that Pgrmc1 function is likely to impact diverse neural functions.

Transcription initiates when the enzyme binds to the promoter region and melts open an initial transcribing bubble that extends from position -4 to +4. The initiation phase continues until the enzyme synthesizes ∼8mer RNA, at which point T7 RNA polymerase begins its transition into the elongation phase. The initiation phase is characterized by a energetic instability, which leads to release of small RNA 2-8 bases in length, known as abortive cycling. Abortive cycling, which is the release of small RNA transcripts during synthesis of the first 8 bases of a transcript, has been well documented in most single and multisubunit RNA polymerases, and has been shown to occur in vivo. Structural studies have prompted the ‘scrunched intermediate’ mechanistic model (an elaboration of the earlier stressed intermediate model), which proposes that compaction of the upstream template DNA within the enzyme and/or expansion of the bubble during initiation leads to instability and the release of abortive RNAs. T7 polymerases represent one of the most well characterized transcription systems and despite having no structural similarities to other multi subunit polymerases, shares very similar fundamental mechanistic features. In the initially transcribing abortive phase, the bubble expands as the initial RNA:DNA hybrid grows and the hybrid pushes on components of the enzyme: both key features in the proposed scrunching mechanism. In this work, we directly test predictions of the scrunching model. The introduction of nicks or gaps into the template (scrunched) strand should reduce stress and therefore reduce abortive. Similarly, the introduction of extra bases in this region should increase the release of abortive RNAs or shift their profile to shorter lengths. For all of these modifications, our results show no systematic change in the abortive amounts or profile. An alternate model predicts that a critical source of stress during abortive initiation is the stress caused by the steric clash of the DNA-RNA hybrid against the N-terminal domain. It is already known that this steric clash leads to the transition of the enzyme into a stable elongation complex, however it is unclear as to whether it induces energetic instability within the system. By introducing bulk at the 5’ end of an initiating RNA primer we have increased the putative stress of the DNA-RNA primer against the N terminal domain and demonstrated a slight increase in the release of abortive products. The increase in abortive RNA was however not systematic and even with an increase in the steric push, the enzyme continues to transition into the elongation phase and make run off RNA. Our results suggest that abortive cycling is a kinetically controlled process as opposed to any structurally mediated mechanism.

Skeletal muscle is dynamic and actively regenerates following damage or altered functional demand. Regeneration is essential for the maintenance of muscle mass and, when dysregulated as a result of disease or aging, can lead to losses in functional capacity and increased mortality. Limited data exist on the molecular mechanisms that govern skeletal muscle regeneration in humans. Therefore, the overall objective of this dissertation was to characterize early molecular alterations in human skeletal muscle to strenuous exercise known to induce a muscle regenerative response. Thirty-five subjects completed 100 eccentric (muscle lengthening) contractions (EC) of the knee extensors with one leg and muscle biopsies were taken from both legs 3 h post-EC. The sample from the non-EC leg served as the control. A well-powered transcriptomic screen and network analysis using Ingenuity Pathway software was first conducted on mRNA from the biopsy samples. Network analysis identified the transcription factor NF-kappaB (NF-kB) as a key molecular element affected by EC. Conformational qRT-PCR confirmed alterations in genes associated with NF-kappaB. A transcription factor ELISA, using nuclear extracts from EC and control muscle samples showed a 1.6 fold increase in NF-kB DNA binding activity following EC. Immunohistochemical experiments then localized the majority of NF-kB positive nuclei to cells in the interstitium, which stained positive for markers of pericyte cells and not satellite cells. To ascertain the mechanistic significance of NF-kB activation following muscle damage, in vitro analyses were carried out using a novel primary pericyte/myoblast co-culture model. Primary pericyte/myoblast co-culture experiments demonstrated that pericytes, transfected with a DNA vector designed to drive NF-kB activation, enhanced proliferation and inhibited myogenic differentiation of co-cultured skeletal muscle myoblasts. Furthermore, reduced NF-kB activation led to enhanced myogenic potential of primary pericytes. Taken together, the data in this dissertation suggest that NF-kB dependent signaling in pericytes regulates myogenic differentiation in a cell- and non-cell autonomous manner and may affect the early regenerative response following muscle damage by inhibiting differentiation and promoting proliferation of muscle satellite cells.

Hsp70 molecular chaperones protect proteins from aggregation, assist in their native structure formation, and regulate stress responses in the cell. A mechanistic understanding of Hsp70 function will be necessary to explain its physiological roles and guide the therapeutic modulation of various disease states. To this end, several fundamental features of the Hsp70 structure-function relationship are investigated. The central component of Hsp70 chaperone function is its capacity for allosteric signaling between structural domains and tunable binding of misfolded protein substrates. In order to identify a cooperative network of sites that mediates interdomain allostery within Hsp70, a mutational correlation analysis is performed using genetic data. Evolutionarily correlations that describe an allosteric network are validated by examining roles for implicated sites in cellular fitness and molecular function. In a second component of the Hsp70 molecular mechanism, a novel function is discovered for the disordered C-terminal tail. This region of the protein enhances the refolding efficiency of substrate proteins independently of interdomain allostery and is required in the cell upon depletion of compensatory chaperones, suggesting a previously undescribed mode of chaperone action. Finally, experiments are initiated to assess the dynamic assembly of Hsp70 domains in various allosteric states and how domain orientations may be guided through interaction with partner co-chaperone proteins.

Through their motion, proteins perform essential functions in the living cell. Although we cannot observe protein motion directly, over 68,000 crystal structures are freely available from the Protein Data Bank. Computational protein rigidity analysis systems leverage this data, building a mechanical model from atoms and pairwise interactions determined from a static 3D structure. The rigid and flexible components of the model are then calculated with a pebble game algorithm, predicting a protein's flexibility with much more computational efficiency than physical simulation. In prior work with rigidity analysis systems, the available modeling options were hard-coded, and evaluation was limited to case studies. The focus of this thesis is improving accuracy and robustness of rigidity analysis systems. The first contribution is in new approaches to mechanical modeling of noncovalent interactions, namely hydrogen bonds and hydrophobic interactions. Unlike covalent bonds, the behavior of these interactions varies with their energies. I systematically investigate energy-refined modeling of these interactions. Included in this is a method to assign a score to a predicted cluster decomposition, adapted from the B-cubed score from information retrieval. Another contribution of this thesis is in new approaches to measuring the robustness of rigidity analysis results. The protein's fold is held in place by weak, noncovalent interactions, known to break and form during natural fluctuations. Rigidity analysis has been conventionally performed on only a single snapshot, rather than on an entire trajectory, and no information was made available on the sensitivity of the clusters to variations in the interaction network. I propose an approach to measure the robustness of rigidity results, by studying how detrimental the loss of a single interaction may be to a cluster's rigidity. The accompanying study shows that, when present, highly critical interactions are concentrated around the active site, indicating that nature has designed a very versatile system for transitioning between unique conformations. Over the course of this thesis, we develop the KINARI library for experimenting with extensions to rigidity analysis. The modular design of the software allows for easy extensions and tool development. A specific feature is the inclusion of several modeling options, allowing more freedom in exploring biological hypotheses and future benchmarking experiments.

Removal of protein activity by genetic mutation or pharmacological inhibition has been used extensively to understand the normal function of a protein. However, null mutations eliminate gene function in all cells and pharmacological agents can diffuse through tissues to have similar global effects that can obscure the physiological function of a protein. This is a particular problem when studying proteins that function in many cell types or that have different cell-specific activities. The most direct strategy to study the function of a protein is to reduce or eliminate its activity only in specific cell types, rather than in all cells of an organism. The idea of targeting gene knockdown to specific cell types or to individual cells is not new and many strategies aim to do just this. However, these strategies result in variable knockdown efficiencies and can have silencing effects in neighboring cells and therefore knockdown is never cell-specific. We developed a novel method to knock down the expression of any gene and to restrict this knockdown to specific cell types in C. elegans. In this method we replaced endogenous genes with single copy integrated transgenes containing an engineered sequence tag that introduces premature stop codons (PTCs) into transgene mRNA. This tag causes the natural stop codon to be recognized as a PTC by the host's nonsense-mediated decay (NMD) machinery and does not disrupt gene function. In NMD-competent animals, a PTC-containing transgene is degraded and in NMD-defective animals, a PTC-containing transgene is expressed. Therefore, the expression of PTC-containing transgenes can be controlled by cell-specific activation of NMD. Using this technique, we replaced two endogenous genes with PTC-containing transgenes and directed degradation of their mRNA to specific cell types by restoring NMD activity in these cells. The single copy transgenes were expressed at levels comparable to the endogenous genes and were knocked down to ∼10% of endogenous by NMD, resulting in both global and cell-specific null-like phenotypes. This knockdown strategy can be used to cell-specifically knock down essentially any gene in the C. elegans genome and should provide new insights into understanding protein function.

During the course of normal DNA replication, replication forks are constantly encountering "housekeeping" types of routine damage to the DNA template that may cause the forks to stall or collapse. One product of this fork collapse is the induction of the SOS response, a coordinated global response to help pause the growth and replication of a cell while DNA damage is addressed and repaired. In E. coli, this response is activated by the formation of ssDNA, to which the RecA protein binds and forms a nucleoprotein filament, which acts as the activator for autocleavage of the LexA transcriptional repressor, which normally represses expression of SOS genes. Damage responses are crucial to maintaining genomic integrity, and are therefore essential to all forms of life, and this type of regulatory system is highly conserved. However, cells have mechanisms for tightly regulating induction of these responses, and can often repair routine damage to their chromosomes without the need to induce SOS. This is chiefly evidenced by the observation that more than 20% of cells in a population have RecA filaments, but less than 1% are induced for SOS. How cells make this decision to induce SOS is the subject of this work. This dissertation describes three projects aimed at examining molecular mechanisms by which cells regulate RecA filaments, and therefore the decision to induce the SOS response. The first examines the disparity between the formation of RecA filaments, as evidenced by RecA-GFP foci, and the induction of SOS in the absence of damage, using a psulA-gfp reporter system. It is shown that there are three independent factors that repress SOS expression in undamaged E. coli cells. These are radA, the amount of recA in the cell, and in some circumstances recX. The first two limit SOS in wild type cells in the absence of external damage, while the third is an additional factor required in xthA mutants, likely due to the fact there are more RecA loading events in these mutants. These factors are thought to change the character and reduce the half-life and persistence of RecA filaments in the cell. The second project shows that suppression of SOS through the use of recA4162 and uvrD303 mutants is substrate and situation-specific. This specificity is demonstrated by the fact that, while both recA4162 and uvrD303 can suppress SOS in the SOS constitutive mutant recA730, recA4162 can only suppress SOS when the signal occurs at replication forks and not at any other place on the chromosome, while uvrD303 appears to suppress SOS with less specificity, and can suppress after UV (shown previously), at induced DSBs, and other places not directly at the replication fork. Here mutants of different replication factors are used that uncouple the replisome and induce SOS to a high degree. The third project determines the factors necessary for loading RecA filaments at the replication fork versus other locations on the chromosome when SOS is induced in the absence of damage, and helps elucidate further mechanisms for induction of SOS at these substrates. It is shown that the sbcB and recJ exonucleases assist in inappropriate RecA filament formation by substrate processing exclusively at replication forks, but not other substrates, likely through mechanisms that are reliant on the activities of the RecA loading factors RecBCD and RecFOR.

Lysosomal storage diseases are disorders caused by deficiencies of enzymes responsible for the degradation of substances present in lysosomes. The loss of activity of a lysosomal enzyme leads to the accumulation of substrates within the lysosome, which is the initial step in the process leading to a lysosomal storage disease. Over fifty lysosomal storage diseases are known and have a collective incidence of approximately 1 in 7700 live births. One treatment for these diseases is enzyme replacement therapy, where the defective enzyme is replaced by a recombinant enzyme. Pharmacological chaperone therapy is an alternative treatment which uses small molecule inhibitors to stabilize the defective enzymes. The purpose of this project is to extend the study of lysosomal storage diseases into the field of molecular medicine by exploring the structure and function of human lysosomal enzymes, specifically those responsible for three variants of the mucopolysaccharidosis (MPS) family of diseases. These three enzymes fall into two categories: N-acetylgalactosamine-6-sulfatase (GALNS) and N-sulfoglucosamine sulfohydrolase (SGSH) are human lysosomal sulfatases while β-galactosidase (GLB1) is a glycosidase. In order to accomplish this goal we used a biochemical and structural biology approach. We solved the structures of SGSH and GALNS using X-ray crystallography and analyzed the mutations that lead to their respective diseases MPS III A and MPS IV A. This analysis revealed that these diseases could reflect protein misfolding since the majority of the mutations is located in the hydrophobic core of the proteins. The active site pockets of these enzymes have features such as charged amino acids and hydrophobic amino acids suitable for drug design. We determined that the small molecules galactose and 1-deoxygalactonojirimycin (DGJ) and 4-epi-isofagomine are competitive inhibitors of GLB1. Using the published crystal structure of GLB1, we present an explanation for the inhibitory effects of 4-epi-isofagomine.Through the structural analysis of the disease causing mutations and the identification of novel inhibitors; we hope to gain insight into the enzymatic mechanisms of these enzymes and the potential candidates for pharmacological chaperone therapy as treatments for the MPS family of diseases.

Incorporation of aryl bromide functionality into proteins was achieved via engineering the bacterial biosynthetic apparatus. A phenylalanine auxotrophic E. coli host was equipped with a phenylalanyl-tRNA synthetase (PheRS) variant that has a broadened substrate specificity. The mutant pheS gene (pheS*), which codes for the α-subunit of the enzyme PheRS and confers relaxed substrate specificity, was encoded on a multiple-copy plasmid that also bears the target gene dihydrofolate reductase (DHFR). Constitutive over-expression of pheS* and subsequent expression of the target gene in the presence of phenylatanine analog, para-bromophenylalanine (p-Br-phe), allowed over 85% replacements of phe residues by p-Br-phe in DHFR. The level of bromination can be controlled by varying the relative amounts of phe and p-Br-phe in the culture medium. Introduction of aryl bromide functionality into proteins offers great potential for selective chemical modification of proteins via transition metal-catalyzed reactions, which are orthogonal to existing protein chemistry. Moreover, bromination may be useful in X-ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique. The utility of the aryl bromide as a unique functionality was investigated in collaboration with Isaac Carrico. Artificial extracellular matrix (ECM) proteins were synthesized using the principles of recombinant DNA technology. These proteins were designed for eventual application in vascular grafts. The engineered ECM proteins contained alternating blocks of cell-binding domains derived from CS1or CS5 regions of human fibronectin for endothelial cell attachment, and elastin-like repeats for mechanical integrity. One phe residue per elastin block [(VPGVG)2VPGFG(VPGVG)2] was designed, which could be replaced with p-Br-phe and subsequently used for chemical cross-linking of the proteins. Protein expression yields of 75–90 mg/L were obtained with 50–60% substitution of phe by p-Br-phe. Preliminary exploration of Pd(0)-catalyzed Heck and Sonagashira couplings with p-Br-phe demonstrate feasibility of these reactions under mild conditions required for protein modification as well as compatibility with side chains of all natural amino acids (except cysteine). Site-specific incorporation of p-Br-phe was tested in an E. coli strain equipped with a yeast PheRS/tRNA Phe amber supressor pair. While p-F-phe can be site-specifically incorporated using this system, attempts at p-Br-phe incorporation were unsuccessful, probably due to unfavorable interaction of p-Br-phe with the bulky and polar tyrosine residue in the binding pocket of yeast PheRS.

U14 small nucleolar RNA (snoRNA) is required for processing of 18S ribosomal RNA. It was hypothesized that U14 might base pair with 18S RNA through two highly conserved U14 sequence elements known as domains A and B. Using Saccharomyces cerevisiae as the experimental system, I showed that: (1) the domain A and B elements are functionally interdependent, and (2) single-point mutations in domain A combined with complete substitution of domain B causes lethality while either mutation alone does not. Direct interaction of U14 with 18S RNA was shown by demonstrating that a lethal mutation in U14 domain A can be suppressed with a mutation which restores complementarity in the corresponding region of 18S RNA. Y-domain in yeast U14 was postulated to serve as a recognition element for vital intermolecular or intramolecular interactions. Consistent with this assumption, mutations in several conserved nucleotides of the loop cause growth defects. In contrast, alterations to the stem have little or no effect. Using a lethal mutation in the loop, three different intragenic suppressor mutations were mapped to three positions adjacent to the primary mutation, and are predicted to influence the structure of the loop. An extragenic suppressors (UF1) able to rescue a cold-sensitive mutation in the loop encodes an essential putative ATP-dependent RNA helicase. Loss of UF1 gene expression caused a reduction in 18S rRNA production, without affecting accumulation of 25S rRNA or U14 snoRNA. Pulse-chase analysis showed that depletion of UF1 protein impaired pre-18S rRNA processing. Finally, an effort was made to define minimum pre-rRNA substrates that can be used to produce functional 18S and 25S rRNAs in vivo. The rDNA operon was split either between the 18S RNA and 5.8S/25S coding units, or between the 18S/5.8S RNA and 25S RNA coding units. The test fragments were expressed from GAL7 promoters. The results showed that functional rRNAs could be produced in trans, but only when the operon was divided between the 18S RNA and 5.8S/25S RNA coding sequence.