Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channel Nav1.7 has gained traction as a pain target with recognition that loss-of-function mutations in SCN9A, the gene encoding Nav1.7, are associated with congenital insensitivity to pain, whereas gain-of-function mutations produce distinct pain syndromes due to increased Nav1.7 activity. Selective inhibition of Nav1.7 is fundamental to modulating pain via this channel. Understanding the regulation of Nav1.7 at the cellular and molecular level is critical for advancing better therapeutics for pain. Although trafficking of Nav1.7 remains poorly understood, recent studies have begun to investigate post-translational modifications of Navs and/or auxiliary subunits as well as protein-protein interactions as Nav-trafficking mechanisms. Here, I tested if post-translational modifications of a novel Nav1.7-interacting protein, the axonal collapsin response mediator protein 2 (CRMP2) by small ubiquitin-like modifier (SUMO) and phosphorylation could affect Nav trafficking and function. Expression of a CRMP2 SUMOylation incompetent mutant (CRMP2-K374A) in neuronal model CAD cells, which express predominantly Nav1.7 currents, led to a significant reduction in huwentoxin-IV-sensitive Nav1.7 currents. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wildtype CRMP2-expressing CAD cells decreased Nav1.7 currents. Consistent with reduced current density, biotinylation revealed significant reduction in surface Nav1.7 levels of CAD cells expressing CRMP2-K374A or SENP proteins. Diminution of Nav1.7 sodium current was recapitulated in sensory neurons expressing CRMP2-K374A. Because CRMP2 functions are regulated by its phosphorylation state, I next investigated possible interplay between phosphorylation and SUMOylation of CRMP2 on Nav1.7. Phosphorylation of CRMP2 by cyclin dependent kinase 5 (Cdk5) was necessary for maintaining Nav1.7 surface expression and current density whereas phosphorylation by Fyn kinase reduced CRMP2 SUMOylation and Nav1.7 current density. Binding to Nav1.7 was decreased following (i) loss of CRMP2 SUMOylation, (ii) loss of CRMP2 phosphorylation by Cdk5, or (iii) gain of CRMP2 phosphorylation by Fyn. Altering CRMP2 modification events simultaneously was not synergistic in reducing Nav1.7 currents, suggesting that Nav1.7 co-opts multiple CRMP2 modifications for regulatory control of this channel. Loss of either CRMP2 SUMOylation or Cdk5 phosphorylation triggered Nav1.7 internalization involving E3 ubiquitin ligase Nedd4-2 as well as endocytosis adaptor proteins Numb and Eps15. Collectively, my findings identify a novel mechanism for regulation of Nav1.7.

Post-translational modifications (PTMs) of proteins can be a sign and/or cause of disease. These modified proteins have the potential to be used as biomarkers for diagnostic purposes. However, research in the field is limited. The challenge of having an accessible way of diagnosing patients in time and at a low cost is crucial to improve public health. Blood samples or other non-invasive methods to detect diseases such as Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis and cancers are of urgent need. This report investigates PTMs as possible biomarkers measurable in biofluids, such as blood, for diagnosis and prognosis. Biomarkers like phospho-tau and amyloid-beta are examined in the context of neurodegenerative diseases, as well as phosphorylations on neurofilaments, TAR DNA-binding protein 43 and α-synuclein. All of these are detectable in blood. Several PTMs with connection to different types of cancers are also investigated, such as F3-phosphopeptide and AFP-L3. It was found that many biomarkers for the detection of cancers can potentially be found in extracellular vesicles in blood. Methods such as ELISA, PEA, SomaScan, xMAP, SIMOA and mass spectrometry (MS) are all now available on the market to quantify these PTMs. MS has revolutionized the fields of protein detection in the past and has further evolved to being capable of protein quantification. ELISA has been prevalent for decades and laid the groundwork for improved methods such as xMAP and SIMOA that are easy to use and provide adequate sensitivity. SomaScan and PEA lead the way in dynamic range and multiplexing capacity with around 7000 and 3000 protein assays. The soon-to-be-released technology NULISA, with promising values in sensitivity and dynamic range, is also investigated here. Additionally, a written ethical analysis regarding the process and consequences of biomarker quantification through these technologies was performed. Although the investigated biomarkers are detectable in biofluids, using them as clinical diagnostic markers still poses a challenge, which is why further research in the field is needed. Through an increased knowledge of PTMs of proteins and the right use of platforms, clinical diagnostics and population screenings can be done more efficiently improving public health around the world.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

ARF, a potent tumor suppressor, positively regulates p53 by antagonizing MDM2, a negative regulator of p53, which in turn, results in either apoptosis or cell cycle arrest. ARF also suppresses the proliferation of cells lacking p53, and loss of ARF in p53-null mice, compared with ARF-null or p53-null mice, results in a broadened tumor spectrum and decreased tumor latency. This evidence suggests that ARF exerts both p53-dependent and p53-independent tumor suppressor activity. However, the molecular pathway and mechanism of ARF’s p53-independent tumor suppressor activity is not understood. The antiapoptotic, metabolically regulated, transcriptional corepressor C-terminal binding protein 2 (CtBP2) has been identified as a specific target of ARF’s p53-independent tumor suppression. CtBPs are phosphoproteins with PLDLS-binding motif and NADH-binding central dehydrogenase domains. ARF interacts with CtBP1 and CtBP2 both in vitro and in vivo, and induces their proteasome-mediated degradation, resulting in p53-independent apoptosis in colon cancer cells. ARF’s ability to target CtBP2 for degradation, and its induction of p53-independent apoptosis requires an intact interaction with CtBP2, and phosphorylation at S428 of CtBP2. As targets for inhibition by ARF, CtBPs are candidate oncogenes, and their expression is elevated in a majority of human colorectal adenocarcinomas specimens in comparison to normal adjacent tissue. Relevant to its targeting by ARF, there is an inverse correlation between ARF and CtBP expression, and CtBP2 is completely absent in a subset of colorectal adenocarcinomas that retains high levels of ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, and they repress epithelial and proapoptotic genes. BH3-only genes such as Bik, Bim and Bmf have been identified as mediators of ARF-induced, CtBP2-mediated p53-indpendent apoptosis. CtBP2 repressed BH3-only genes in a tissue specific manner through BKLF (Basic kruppel like factor)-binding elements. ARF regulation of BH3-only genes also required intact interaction with CtBP2. ARF antagonism of CtBP repression of Bik and other BH3-only genes may play a critical role in ARF-induced p53-independent apoptosis, and in turn, tumor suppression. To study the physiologic effect of ARF/CtBP2 interaction at the organismal level, the p19ArfL46D knock-in mice, in which the Arf/CtBP2 interaction was abrogated, was generated. Analysis of the primary cells derived from these mice, revealed that the Arf/CtBP2 interaction contributes to regulation of cell growth and cell migration. Overexpression of CtBP in human tumors, and ARF antagonism of CtBP repression of BH3-only gene expression and CtBP-mediated cell migration may therefore play a critical role in the p53-independent tumor suppressor function/s of ARF.

Indiana University-Purdue University Indianapolis (IUPUI) / 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.

No / During development, multipotent progenitor cells establish tissue-specific programs of gene expression. In this paper, we show that p63 transcription factor, a master regulator of epidermal morphogenesis, executes its function in part by directly regulating expression of the genome organizer Satb1 in progenitor cells. p63 binds to a proximal regulatory region of the Satb1 gene, and p63 ablation results in marked reduction in the Satb1 expression levels in the epidermis. Satb1(-/-) mice show impaired epidermal morphology. In Satb1-null epidermis, chromatin architecture of the epidermal differentiation complex locus containing genes associated with epidermal differentiation is altered primarily at its central domain, where Satb1 binding was confirmed by chromatin immunoprecipitation-on-chip analysis. Furthermore, genes within this domain fail to be properly activated upon terminal differentiation. Satb1 expression in p63(+/-) skin explants treated with p63 small interfering ribonucleic acid partially restored the epidermal phenotype of p63-deficient mice. These data provide a novel mechanism by which Satb1, a direct downstream target of p63, contributes in epidermal morphogenesis via establishing tissue-specific chromatin organization and gene expression in epidermal progenitor cells.

Thymic epithelial cells (TECs) are the main component of the thymic stroma, which supports T-cell proliferation and repertoire selection. Here, we demonstrate that Cbx4, a Polycomb protein that is highly expressed in the thymic epithelium, has an essential and non-redundant role in thymic organogenesis. Targeted disruption of Cbx4 causes severe hypoplasia of the fetal thymus as a result of reduced thymocyte proliferation. Cell-specific deletion of Cbx4 shows that the compromised thymopoiesis is rooted in a defective epithelial compartment. Cbx4-deficient TECs exhibit impaired proliferative capacity, and the limited thymic epithelial architecture quickly deteriorates in postnatal mutant mice, leading to an almost complete blockade of T-cell development shortly after birth and markedly reduced peripheral T-cell populations in adult mice. Furthermore, we show that Cbx4 physically interacts and functionally correlates with p63, which is a transcriptional regulator that is proposed to be important for the maintenance of the stemness of epithelial progenitors. Together, these data establish Cbx4 as a crucial regulator for the generation and maintenance of the thymic epithelium and, hence, for thymocyte development.

A cellular pre-mRNA undergoes various post-transcriptional processing events, including capping, splicing and polyadenylation prior to nuclear export. Splicing is particularly important for mRNA nuclear export as two distinct multi-protein complexes, known as human TREX (hTREX) and the exon-junction complex (EJC), are recruited to the mRNA in a splicing-dependent manner. In contrast, a number of Kaposi's sarcoma-associated herpesvirus (KSHV) lytic mRNAs lack introns and are exported by the virus-encoded ORF57 protein. Herein we show that ORF57 binds to intronless viral mRNAs and functions to recruit the complete hTREX complex, but not the EJC, in order assemble an export component viral ribonucleoprotein particle (vRNP). The formation of this vRNP is mediated by a direct interaction between ORF57 and the hTREX export adapter protein, Aly. Aly in turn interacts directly with the DEAD-box protein UAP56, which functions as a bridge to recruit the remaining hTREX proteins to the complex. Moreover, we show that a point mutation in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in the ORF57-mediated recruitment of the entire hTREX complex to the intronless viral mRNA and inhibits the mRNAs subsequent nuclear export and virus replication. Furthermore, we have utilised a trans-dominant Aly mutant to prevent the assembly of the complete ORF57-hTREX complex; this results in a vRNP consisting of viral mRNA bound to ORF57, Aly and the nuclear export factor, TAP. Strikingly, although both the export adapter Aly and the export factor TAP were present on the viral mRNP, a dramatic decrease in intronless viral mRNA export and virus replication was observed in the absence of the remaining hTREX components (UAP56 and hTHO-complex). Together, these data provide the first direct evidence that the complete hTREX complex is essential for the export of KSHV intronless mRNAs and infectious virus production.