JIP scaffold proteins are implicated in the regulation of protein kinase signal transduction pathways. To test the physiological role of these scaffold proteins, we examined the phenotype of compound mutant mice that lack expression of JIP proteins. These mice were found to exhibit severe defects in N-methyl-D-aspartic acid (NMDA) receptor function, including decreased NMDA-evoked current amplitude, cytoplasmic Ca(++), and gene expression. The decreased NMDA receptor activity in JIP-deficient neurons is associated with reduced tyrosine phosphorylation of NR2 subunits of the NMDA receptor. JIP complexes interact with the SH2 domain of cFyn and may therefore promote tyrosine phosphorylation and activity of the NMDA receptor. We conclude that JIP scaffold proteins are critically required for normal NMDA receptor function.
The regulation of innate immune responses to pathogens occurs through the interaction of Toll-like receptors (TLRs) with pathogen-associated molecular patterns and the activation of several signaling pathways whose contribution to the overall innate immune response to pathogens is poorly understood. We demonstrate a mechanism of control of murine macrophage responses mediated by TLR1/2 heterodimers through c-Jun N-terminal kinase 1 (JNK1) activity. JNK controls tumor necrosis factor alpha production and TLR-mediated macrophage responses to Borrelia burgdorferi, the causative agent of Lyme disease, and the TLR1/TLR2-specific agonist PAM(3)CSK(4). JNK1, but not JNK2, activity regulates the expression of the tlr1 gene in the macrophage cell line RAW264.7, as well as in primary CD11b(+) cells. We also show that the proximal promoter region of the human tlr1 gene contains an AP-1 binding site that is subjected to regulation by the kinase and binds two complexes that involve the JNK substrates c-Jun, JunD, and ATF-2. These results demonstrate that JNK1 regulates the response to TLR1/2 ligands and suggest a positive feedback loop that may serve to increase the innate immune response to the spirochete.
Activity-Based Profiling Reveals a Regulatory Link between Oxidative Stress and Protein Arginine Phosphorylation
Protein arginine phosphorylation is a recently discovered modification that affects multiple cellular pathways in Gram-positive bacteria. In particular, the phosphorylation of arginine residues by McsB is critical for regulating the cellular stress response. Given that the highly efficient protein arginine phosphatase YwlE prevents arginine phosphorylation under non-stress conditions, we hypothesized that this enzyme negatively regulates arginine phosphorylation and acts as a sensor of cell stress. To evaluate this hypothesis, we developed the first suite of highly potent and specific SO3-amidine-based YwlE inhibitors. With these protein arginine phosphatase-specific probes, we demonstrated that YwlE activity is suppressed by oxidative stress, which consequently increases arginine phosphorylation, thereby inducing the expression of stress-response genes, which is critical for bacterial virulence. Overall, we predict that these novel chemical tools will be widely used to study the regulation of protein arginine phosphorylation in multiple organisms.
The Identification and Targeting of Partially-Folded Conformations on the Folding Free-Energy Landscapes of ALS-Linked Proteins for Therapeutic Intervention: A Dissertation
The hallmark feature of many neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the accumulation of cytoplasmic inclusions of key disease-linked proteins. Two of these proteins, TDP-43 and SOD1, represent a significant proportion of sporadic and familial ALS cases, respectively. The population of potentially aggregation-prone partially-folded states on the folding free-energy landscape may serve as a common mechanism for ALS pathogenesis. A detailed biophysical understanding of the folding and misfolding energy landscapes of TDP-43 and SOD1 can provide critical insights into the design of novel therapeutics to delay onset and progression in ALS.
Equilibrium unfolding studies on the RNA recognition motif (RRM) domains of TDP-43 revealed the population of a stable RRM intermediate in RRM2, with residual structure localized to the N-terminal half of the domain. Other RRM domains from FUS/TLS and hnRNP A1 similarly populate RRM intermediates, suggesting a possible connection with disease. Mutations, which enhance the population of the RRM2 intermediate, could serve as tools for deciphering the functional and misfolding roles of this partially-folded state in disease models, leading to the development of new biomarkers to track ALS progression.
ALS mutations in SOD1 have been shown to destabilize the stable homodimer to result in increased populations of the monomeric and unfolded forms of SOD1. Mechanistic insights into the misfolding of SOD1 demonstrated that the unfolded state is a key species in the initiation and propagation of aggregation, suggesting that limiting these populations may provide therapeutic benefit to ALS patients. An in vitro time-resolved Förster Resonance Energy Transfer assay to screen small molecules that stabilize the native state of SOD1 has identified several lead compounds, providing a pathway to new therapeutics to treat ALS.
XIST and CoT-1 Repeat RNAs are Integral Components of a Complex Nuclear Scaffold Required to Maintain SAF-A and Modify Chromosome Architecture: A Dissertation
XIST RNA established the precedent for a noncoding RNA that stably associates with and regulates chromatin, however it remains poorly understood how such RNAs structurally associate with the interphase chromosome territory. I demonstrate that transgenic XIST RNA localizes in cis to an autosome as it does to the inactive X chromosome, hence the RNA recognizes a structure common to all chromosomes. I reassess the prevalent thinking in the field that a single protein, Scaffold Attachment Factor-A (SAF-A/hnRNP U), provides a single molecule bridge required to directly tether the RNA to DNA. In an extensive series of experiments in multiple cell types, I examine the effects of SAF-A depletion or different SAF-A mutations on XIST RNA localization, and I force XIST RNA retention at mitosis to examine the effect on SAF-A. I find that SAF-A is not required to localize XIST RNA but is one of multiple proteins involved, some of which frequently become lost or compromised in cancer. I additionally examine SAF-A’s potential role localizing repeat-rich CoT-1 RNA, a class of abundant RNAs that we show tightly and stably localize to euchromatic interphase chromosome territories, but release upon disruption of the nuclear scaffold. Overall, findings suggest that instead of “tethering” chromosomal RNAs to the scaffold, SAF-A is one component of a multi-component matrix/scaffold supporting interphase nuclear architecture. Results indicate that Cot-1 and XIST RNAs form integral components of this scaffold and are required to maintain the chromosomal association of SAF-A, substantially advancing understanding of how chromatin-associated RNAs contribute to nuclear structure.
Contraceptive Utilization and Downstream Feto-Maternal Outcomes for Women with Substance Use Disorders: A Dissertation
Background: One in ten people in the U.S. are affected by a substance use disorder (SUD), roughly one third of whom are women. Rates of unintended pregnancy are higher in this population than in the general public. Little is understood about how women with SUD use prescription contraception and think about pregnancy.
Methods: By analyzing Medicaid claims data and conducting qualitative interviews with women with SUD, this doctoral thesis seeks to: 1) compare any use of and consistent, continued coverage by prescription contraceptives between women with and without SUD; 2) determine the extent to which SUD is associated with pregnancy, abortion, and adverse feto-maternal outcomes in women who use prescription contraception; and 3) explore facilitators of and barriers to contraceptive utilization by women with SUD, using qualitative interviews.
Results: Compared to women without SUD, women with SUD are less likely to use any prescription contraceptive, particularly long-acting reversible methods. Among women who do use long-acting methods, SUD is associated with less continued, consistent coverage by a prescription contraceptive. Among women who use contraception, SUD is also associated with increased odds of abortion. When interviewed, women with SUD report fatalistic attitudes towards pregnancy planning, and have difficulty conceptualizing how susceptibility to pregnancy may change over time. Women with SUD also report that pregnancy has substantial impact on their drug treatment prospects.
Conclusions: This study is the first to examine contraceptive utilization by women with SUD who are enrolled in Medicaid or state-subsidized insurance. Our study may help to inform clinical practice and policy development to improve the reproductive health and wellbeing of women with SUD.