Browsing by Subject "Cellular control mechanisms"

Now showing 1 - 10 of 13
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    Development and stability of IL-17-secreting T cells
    (2014) Glosson, Nicole L.; Kaplan, Mark H.; Blum, Janice Sherry, 1957-; Yu, Andy; Harrington, Maureen A.
    IL-17-producing T cells are critical to the development of pathogen and tumor immunity, but also contribute to the pathology of autoimmune diseases and allergic inflammation. CD8+ (Tc17) and CD4+ (Th17) IL-17-secreting T cells develop in response to a cytokine environment that activates Signal Transducer and Activator of Transcription (STAT) proteins, though the mechanisms underlying Tc17/Th17 development and stability are still unclear. In vivo, Tc17 cells clear vaccinia virus infection and acquire cytotoxic potential, that is independent of IL-17 production and the acquisition of IFN-γ-secreting potential, but partially dependent on Fas ligand, suggesting that Tc17-mediated vaccinia virus clearance is through cell killing independent of an acquired Tc1 phenotype. In contrast, memory Th cells and NKT cells display STAT4-dependent IL-23-induced IL-17 production that correlates with Il23r expression. IL-23 does not activate STAT4 nor do other STAT4-activating cytokines induce Il23r expression in these populations, suggesting a T cell-extrinsic role for STAT4 in mediating IL-23 responsiveness. Although IL-23 is important for the maintenance of IL-17-secreting T cells, it also promotes their instability, often resulting in a pathogenic Th1-like phenotype in vitro and in vivo. In vitro-derived Th17 cells are also flexible when cultured under polarizing conditions that promote Th2 or Th9 differentiation, adopting the respective effector programs, and decreasing IL-17 production. However, in models of allergic airway disease, Th17 cells do not secrete alternative cytokines nor adopt other effector programs, and remain stable IL-17-secretors. In contrast to Th1-biased pro-inflammatory environments that induce Th17 instability in vivo, during allergic inflammatory disease, Th17 cells are comparatively stable, and retain the potential to produce IL-17. Together these data document that the inflammatory environment has distinct effects on the stability of IL-17-secreting T cells in vivo.
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    Functional Insights Into Oncogenic Protein Tyrosine Phosphatases By Mass Spectrometry
    (2012-12) Walls, Chad Daniel; Zhang, Zhong-Yin; Wang, Mu; Wells, Clark D.; Zhang, Jian-Ting
    Phosphatase of Regenerating Liver 3 (PRL3) is suspected to be a causative factor toward cellular metastasis when overexpressed. To date, the molecular basis for PRL3 function remains an enigma, justifying the use of 'shot-gun'-style phosphoproteomic strategies to define the PRL3-mediated signaling network. On the basis of aberrant Src tyrosine kinase activation following ectopic PRL3 expression, phosphoproteomic data reveal a signal transduction network downstream of a mitogenic and chemotactic PDGF (α and β), Eph (A2, B3, B4), and Integrin (β1 and β5) receptor array known to be utilized by migratory mesenchymal cells during development and acute wound healing in the adult animal. Tyrosine phosphorylation is present on a multitude of signaling effectors responsible for Rho-family GTPase, PI3K-Akt, Jak-STAT3, and Ras-ERK1/2 pathway activation, linking observations made by the field as a whole under Src as a primary signal transducer. Our phosphoproteomic data paint the most comprehensive picture to date of how PRL3 drives pro-metastatic molecular events through Src activation. The Src-homology 2 (SH2) domain-containing tyrosine phosphatase 2 (SHP2), encoded by the Ptpn11 gene, is a bona-fide proto-oncogene responsible for the activation of the Ras/ERK1/2 pathway following mitogen stimulation. The molecular basis for SHP2 function is pTyr-ligand-mediated alleviation of intramolecular autoinhibition by the N-terminal SH2 domain (N-SH2 domain) upon the PTP catalytic domain. Pathogenic mutations that reside within the interface region between the N-SH2 and PTP domains are postulated to weaken the autoinhibitory interaction leading to SHP2 catalytic activation in the open conformation. Conversely, a subset of mutations resides within the catalytic active site and cause catalytic impairment. These catalytically impaired SHP2 mutants potentiate the pathogenesis of LEOPARD-syndrome (LS), a neuro-cardio-facial-cutaneous (NCFC) syndrome with very similar clinical presentation to related Noonan syndrome (NS), which is known to be caused by gain-of-function (GOF) SHP2 mutants. Here we apply hydrogen-deuterium exchange mass spectrometry (H/DX-MS) to provide direct evidence that LS-associated SHP2 mutations which cause catalytic impairment also weaken the autoinhibitory interaction that the N-SH2 domain makes with the PTP domain. Our H/DX-MS study shows that LS-SHP2 mutants possess a biophysical property that is absolutely required for GOF-effects to be realized, in-vivo.
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    The influence of the Ku80 carboxy-terminus on activation of the DNA-dependent protein kinase and DNA repair is dependent on the structure of DNA cofactors
    (2013-11) Woods, Derek S.; Turchi, John J.; Harrington, Maureen A.; Malkova, Anna L.; Takagi, Yuichiro
    In mammalian cells DNA double strand breaks (DSBs) are highly variable with respect to sequence and structure all of which are recognized by the DNA- dependent protein kinase (DNA-PK), a critical component for the resolution of these breaks. Previously studies have shown that DNA-PK does not respond the same way to all DSBs but how DNA-PK senses differences in DNA substrate sequence and structure is unknown. Here we explore the enzymatic mechanism by which DNA-PK is activated by various DNA substrates. We provide evidence that recognition of DNA structural variations occur through distinct protein-protein interactions between the carboxy terminal (C-terminal) region of Ku80 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs). Discrimination of terminal DNA sequences, on the other hand, occurs independently of Ku 80 C-terminal interactions and results exclusively from DNA-PKcs interactions with the DNA. We also show that sequence differences in DNA termini can drastically influence DNA repair through altered DNA-PK activation. Our results indicate that even subtle differences in DNA substrates influence DNA-PK activation and ultimately Non-homologous End Joining (NHEJ) efficiency.
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    Investigating the early events in proteasome assembly
    (2014) Ramamurthy, Aishwarya; Kusmierczyk, Andrew; Atkinson, Simon; Randall, Stephen Karl, 1953-
    Proteasome assembly is a rapid and highly sequential process that occurs through a series of intermediates. While the quest to understand the exact process of assembly is ongoing, there remains an incomplete understanding of what happens early on during the process, prior to the involvement of the β subunits. A significant feature of proteasome assembly is the property of proteasomal subunits to self-assemble. While archaeal α and β subunits from Thermoplasma acidophilum can assemble into entire 20S units in vitro, certain α subunits from divergent species have a property to self-assemble into single and double heptameric rings. In this study, we have shown that recombinant α subunits from Methanococcus maripaludis also have a tendency to self-assemble into higher order structures when expressed in E. coli. Using a novel cross-linking strategy, we were able to establish that these higher order structures were double α rings that are structurally similar to a half-proteasome (i.e. an α-β ring pair). Our experiments on M. maripaludis α subunits represent the first biochemical evidence for the orientation of rings in an α ring dimer. We also investigated self-assembly of α subunits in S. cerevisiae and attempted to characterize a highly stable and unique high molecular weight complex (HMWC) that is formed upon co-expression of α5, α6, α7 and α1 in E. coli. Using our cross-linking strategy, we were able to show that this complex is a double α ring in which, at the least, one α1 subunit is positioned across itself. We were also able to detect α1-α1 crosslinks in high molecular weight complexes that are formed when α7 and α1 are co-expressed, and when α6, α7 and α1 are co-expressed in E. coli. The fact that we able to observe α1-α1 crosslinks in higher order structures that form whenever α7 and α1 were present suggests that α1-α1 crosslinks might be able to serve as potential trackers to detect HMWCs in vivo. This would be an important step in determining if these HMWCs represent bona fide assembly intermediates, or dead-end complexes whose formation must be prevented in order to ensure efficient proteasome assembly.
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    Investigation of the action of phosphatase of regenerating liver on PTEN using murine models
    (2014-09) Campbell, Amanda Marie; Zhang, Zhong-Yin; Quilliam, Lawrence; Dong, X. Charlie; Mayo, Lindsey D.
    The addition and removal of phosphate groups is a key regulatory mechanism for many cellular processes. The balance between phosphorylation and dephosphorylation is delicate and must be maintained in order for proper cell functions to be carried out. Protein kinases and phosphatases are the keepers of this balance with kinases adding phosphate groups and phosphatases removing them. As such, mutation and/or altered regulation of these proteins can be the driving factor in disease. Phosphatase of Regenerating Liver (PRL) is a family novel of three dual specificity phosphatases (DSPs) first discovered in the regenerating liver tissue of rats. PRLs have also been shown to act as oncogenes in cell culture and in animal models. However, the physiological substrate and mechanisms of the PRLs are not yet known. Recently, our lab has developed a PRL 2 knockout mouse and found several striking phenotypes all of which correspond to a significant increase in PTEN. We also found that PRL 2 is targetable by small molecular inhibitors that can potentially be used to disrupt tumor growth and spermatogenesis. Furthermore, a PTEN heterozygous mouse model crossed into our PRL 2 knockout line was generated to investigate the relevance of PRL interaction with PTEN in cancer.
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    Mechanisms of translational regulation in the pancreatic β cell stress response
    (2014) Templin, Andrew Thomas; Mirmira, Raghavendra G.; Day, Richard N.; Fueger, Patrick T.; Harrington, Maureen A.; Wek, Ronald C.
    The islet beta cell is unique in its ability to synthesize and secrete insulin for use in the body. A number of factors including proinflammatory cytokines, free fatty acids, and islet amyloid are known to cause beta cell stress. These factors lead to lipotoxic, inflammatory, and ER stress in the beta cell, contributing to beta cell dysfunction and death, and diabetes. While transcriptional responses to beta cell stress are well appreciated, relatively little is known regarding translational responses in the stressed beta cell. To study translation, I established conditions in vitro with MIN6 cells and mouse islets that mimicked UPR conditions seen in diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Chronic exposure of beta cells to proinflammatory cytokines (IL-1 beta, TNF-alpha, IFN-gamma), or to the saturated free fatty acid palmitate, led to changes in global beta cell translation consistent with attenuation of translation initiation, which is a hallmark of ER stress. In addition to changes in global translation, I observed transcript specific regulation of ribosomal occupancy in beta cells. Similar to other privileged mRNAs (Atf4, Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes during the UPR, whereas the mRNA encoding a proinsulin processing enzyme (Cpe) partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5’ untranslated region of mouse Pdx1 (between bp –105 to –280) contained elements that promoted translation under both normal and UPR conditions. In contrast to regulation of translation initiation, deoxyhypusine synthase (DHS) and eukaryotic translation initiation factor 5A (eIF5A) are required for efficient translation elongation of specific stress relevant messages in the beta cell including Nos2. Further, p38 signaling appears to promote translational elongation via DHS in the islet beta cell. Together, these data represent new insights into stress induced translational regulation in the beta cell. Mechanisms of differential mRNA translation in response to beta cell stress may play a key role in maintenance of islet beta cell function in the setting of diabetes.
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    Potential role of histone deacetylases in the development of the chick and murine retina
    (2014-09-04) Saha, Ankita; Belecky-Adams, Teri; Stocum, David L.; Meyer, Jason S.; Atkinson, Simon
    The epigenetic state of any cell is, in part, regulated by the interaction of DNA with nuclear histones. Histone tails can be modified in a number of ways that impact on the availability of DNA to interact with transcriptional complexes, including methylation, acetylation, phosphorylation, ubiquituination, and sumoylation. Histones are acetylated by a large family of enzymes, histone acetyl transferases (HATs), and deacetylated by the histone deacetylases (HDACs). Acetylated histones are generally considered markers of genomic regions that are actively being transcribed, whereas deacetylated and methylated histones are generally markers of regions that are inactive. The goal of the present study was to 1) study the epigenetic state with regard to the presence of euchromatin and heterochromatin in the developing chick and murine retina, 2) study and compare the localization patterns of the classical HDACs in the developing chick and murine retina with respect retinal progenitors and early differentiated cell types 3) to test the hypothesis that overall HDAC activity is required for dividing retinal progenitors to leave the cell cycle and differentiate. Our results showed that the classical HDACs were ubiquitously expressed in the developing chick and murine retinas. Species specific differences as well as stage dependent variations were observed in the localization of the HDACs in the cell types that were studied in the chick and murine retina. Our preliminary results also showed that HDAC inhibition may lead to the inability of the cell types to leave the cell cycle and a subsequent increase in the number of progenitor cells present in the developing chick retina.
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    REGULATION OF CHOP TRANSLATION IN RESPONSE TO eIF2 PHOSPHORYLATION AND ITS ROLE IN CELL FATE
    (2012-05) Palam, Lakshmi Reddy; Wek, Ronald C.; Herring, Brian P.; Harris, Robert A. (Robert Allison), 1939-; Skalnik, David G.
    In response to different environmental stresses, phosphorylation of eukaryotic initiation factor-2 (eIF2) rapidly reduces protein synthesis, which lowers energy expenditure and facilitates reprogramming of gene expression to remediate stress damage. Central to the changes in gene expression, eIF2 phosphorylation also enhances translation of ATF4, a transcriptional activator of genes subject to the Integrated Stress Response (ISR). The ISR increases the expression of genes important for alleviating stress, or alternatively triggering apoptosis. One ISR target gene encodes the transcriptional regulator CHOP whose accumulation is critical for stress-induced apoptosis. In this dissertation research, I show that eIF2 phosphorylation induces preferential translation of CHOP by a mechanism involving a single upstream ORF (uORF) located in the 5’-leader of the CHOP mRNA. In the absence of stress and low eIF2 phosphorylation, translation of the uORF serves as a barrier that prevents translation of the downstream CHOP coding region. Enhanced eIF2 phosphorylation during stress facilitates ribosome bypass of the uORF, and instead results in the translation of CHOP. Stable cell lines were also constructed that express CHOP transcript containing the wild type uORF or deleted for the uORF and each were analyzed for expression changes in response to the different stress conditions. Increased CHOP levels due to the absence of inhibitory uORF sensitized the cells to stress-induced apoptosis when compared to the cells that express CHOP mRNA containing the wild type uORF. This new mechanism of translational control explains how expression of CHOP and the fate of cells are tightly linked to the levels of phosphorylated eIF2 and stress damage.
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    Serum response factor-dependent regulation of smooth muscle gene transcription
    (2014-07-07) Chen, Meng; Herring, B. Paul; Gallagher, Patricia J.; Petrache, Irina; Rhodes, Simon J.; Tune, Johnathan D.
    Several common diseases such as atherosclerosis, post-angioplasty restenosis, and graft vasculopathies, are associated with the changes in the structure and function of smooth muscle cells. During the pathogenesis of these diseases, smooth muscle cells have a marked alteration in the expression of many smooth muscle-specific genes and smooth muscle cells undergo a phenotypic switch from the contractile/differentiated status to the proliferative/dedifferentiated one. Serum response factor (SRF) is the major transcription factor that plays an essential role in coordinating a variety of transcriptional events during this phenotypic change. The first goal of my thesis studies is to determine how SRF regulates the expression of smooth muscle myosin light chain kinase (smMLCK) to mediate changes in contractility. Using a combination of transgenic reporter mouse and knockout mouse models I demonstrated that a CArG element in intron 15 of the mylk1 gene is necessary for maximal transcription of smMLCK. SRF binding to this CArG element modulates the expression of smMLCK to control smooth muscle contractility. A second goal of my thesis work is to determine how SRF coordinates the activity of chromatin remodeling enzymes to control expression of microRNAs that regulate the phenotypes of smooth muscle cells. Using both mouse knockout models and in vitro studies in cultured smooth muscle cells I showed how SRF acts together with Brg1-containing chromatin remodeling complexes to regulate expression of microRNAs-143, 145, 133a and 133b. Moreover, I found that SRF transcription cofactor myocardin acts together with SRF to regulate expression of microRNAs-143 and 145 but not microRNAs-133a and 133b. SRF can, thus, further modulate gene expression through post-transcriptional mechanisms via changes in microRNA levels. Overall my research demonstrates that through direct interaction with a CArG box in the mylk1 gene, SRF is important for regulating expression of smMLCK to control smooth muscle contractility. Additionally, SRF is able to harness epigenetic mechanisms to modulate expression of smooth muscle contractile protein genes directly and indirectly via changes in microRNA expression. Together these mechanisms permit SRF to coordinate the complex phenotypic changes that occur in smooth muscle cells.