Browsing by Subject "Proto-Oncogene Proteins p21(ras)"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item ADAP1 Promotes Latent HIV-1 Reactivation by Tuning the KRAS-ERK-AP-1 Signaling-Transcriptional Axis(December 2021) Ramirez, Nora-Guadalupe Piña; Schoggins, John W.; D'Orso, Iván; Pfeiffer, Julie K.; Alto, NealImmune stimulation fuels cell signaling-transcriptional programs that induce biological responses to eliminate virus-infected cells. Yet, retroviruses that integrate into host cell chromatin, such as HIV-1, co-opt these programs to switch between latent and reactivated states. However, many regulatory mechanisms are still unfolding. As such, here I take advantage of the unique intrinsic reliance HIV-1 has on host cell signaling-transcriptional programs to discover undescribed cell signaling regulators. Specifically, I implemented a functional screening platform, given HIV-1 gene expression relies on CD4+ T cell activation state, to identify host factors modulating CD4+ T cell signaling-transcriptional axes and consequently HIV-1 fate. Among the hits, I focus on ADAP1 (ArfGAP with Dual PH Domains 1), a previously thought neuro-restricted factor, and discover it is an amplifier of select human CD4+ T cell signaling programs. Using physiological models, I characterize ADAP1 expression is low in naïve and memory CD4+ T cells, but largely induced upon immune stimulation where it interacts with the immune signalosome. Using complementary biochemical and cellular assays, I demonstrate ADAP1 directly stimulates the GTPase activity of KRAS to amplify CD4+ T cell signaling through targeted activation of ERK-AP-1 axis. In primary CD4+ T cells which I have genetically ablated ADAP1, I show loss of ADAP1 function blunts gene expression programs in response to stimulation thereby reducing CD4+ T cell expansion and dampening latent HIV-1 reactivation. Supporting the impact of these findings, I propose the reduced CD4+ T cell programs and proliferation upon ADAP1 loss validates Genome-wide Association Studies linking ADAP1 single nucleotide polymorphisms in non-coding enhancers to an altered T lymphocyte count trait, potentially attributed to ADAP1 haploinsufficiency. Through these combined experimental approaches, I was able to define ADAP1 as an unexpected tuner of CD4+ T cell activation programs and co-opted by HIV-1 to escape latency.Item B-Catenin and K-ras Synergize to Form Wilm's Tumor with Concurrent p53 Pathways Modulation(2014-02-04) Hembd, Austin; Clark, Peter; DeGraff, DavidHumans can develop pediatric kidney tumors called Wilm's tumors. If one identifies the specific genes that cause Wilm's tumor, or that concomitantly change expression levels in the tumor tissue, then diagnosis and eventually drug targets for therapy are expedited. Characterizing genetic determinants in the mouse model can help actualize these future therapies. When the genes Kras and βCatenin are overexpressed in a mouse, it develops a renal tumor histologically identical to a human Wilm's tumor. Microarray analysis on mouse tumor tissue showed modulated expression levels of gene targets in the p53 tumor suppressor pathway. Immunohistochemistry stained mice tissue specifically for p53. In tissue with Kras and βcatenin overexpression, p53 staining is positive surrounding the tumor. RT qPCR measured levels of gene expression of p53 pathway associated genes. Combination mutants βCatenin and Kras were compared with controls. This PCR array analysis identified genes, such as cJun, Traf1, and Dapk1, that had significant expression changes in the combination mutant when compared to either mutant individually. The expression is modulated in a nonadditive fashion in Kras + βcatenin mutant tissues, which can explain the phenotype of Wilm's tumor in only double mutant mice. These genes individually represent targets for therapy in the future, and together represent an identifying fingerprint for diagnosis and prediction.Item Code Within Codes: Codon Usage Regulate Protein Expression, Structure, and Function(2018-07-23) Fu, Jingjing; Jiang, Jin; Liu, Yi; Cobb, Melanie H.; Green, Carla B.Most amino acids are encoded by two to six synonymous genetic codons. Synonymous codons are not used with the same frequency in all organisms, and every organism has its own preferred codon usage bias. Codon usage bias has been shown to positively correlate with tRNA abundance, thus optimal codons are thought to be translated more efficiently and accurately. Consistent with this, strong codon usage biases have been shown to be important for the expression of highly expressed genes in different organisms, and codon optimization has been widely used to enhance heterologous protein expression. Therefore, codon usage can be an important determinant in gene expression. In addition, codon usage has been shown to influence translation elongation rate and protein structure by affecting the co-translational folding process in E. coli, fungi, and insects. In addition to its role in regulating protein translation, codon usage also has a major role in determining the level of gene expression through transcriptional and post-transcriptional processes. As such, gene codon usage has been proposed to be a code within the genetic code that can determine both gene expression levels and protein structures and therefore activity. However, the effects of codon usage in multi-tissue organisms, for example, animals and humans, are not clear. In the first part of the thesis, by codon-optimizing open reading frame of Drosophila period gene, I showed that dper codon usage is critical for its circadian clock function. Optimization of dper codon usage resulted in conformational changes of dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER repressor function in the circadian negative feedback loop. In the second part of the thesis, I reported that changing rare codons to common in KRAS increased translation and mRNA levels. Regulation of mRNA levels is a major mechanism affecting KRAS levels, but the effect was not a product of mRNA stability, but instead transcriptional. Moreover, codon usage also had an impact on the structure of KRAS. Thus, the rare codon bias of KRAS effects more aspects of protein production and function than previously appreciated, which has important implications for other rare codon enriched mammalian genes.Item Identification of Oncogenic KRAS-Associated Vulnerabilities in Non-Small Cell Lung Cancer(2016-05-26) Kim, Ji Mi; Lum, Lawrence; White, Michael A.; Fontoura, Beatriz; Cobb, Melanie H.Activating mutations in KRAS are frequently involved in the pathogenesis of non-small cell lung cancer (NSCLC), the disease responsible for the most cancer-related deaths in the US. Despite intensive efforts to develop drugs that directly interfere with KRAS activity over the past decade, no effective inhibitor has been developed. As an alternative, synthetic-lethal therapeutic opportunities are being pursued using large-scale, RNAi-based, functional genomics platforms. We first addressed two major challenges associated with RNAi-based primary synthetic-lethal screens; a prevalent miRNA-like behavior of siRNA and cell line-dependent phenotypic diversity within intra-lineage KRAS-driven cancer. In consideration of these, we performed a whole-genome synthetic-lethal siRNA screen, powered by 106 NSCLC lines and integrated with gene set enrichment analysis. This identified components of nuclear transport machinery as selectively essential for KRAS mutant NSCLC lines. We found that pharmacological inhibition of a key nuclear export receptor, XPO1 (a.k.a. CRM1), was sufficient to induce robust and selective apoptosis in KRAS mutant NSCLC cells in vitro and to cause significant impairment of KRAS mutant tumor growth in vivo. Mechanistically, XPO1-depedent nuclear export machinery was required to maintain NFκB-mediated survival signaling. We discovered that a subset of KRAS mutant NSCLC lines bypassed the addiction to XPO1-dependent nuclear export via YAP1 activation as a consequence of previously unappreciated co-occurring loss-of-function mutations in FSTL5 and mutations in Hippo pathway. The intrinsic resistance was reversed by coadministration of YAP1/TEAD inhibitor. Thus, our study suggests that XPO1 can be exploited for a promising therapeutic opportunity for KRAS mutant lung cancer and provides strategies for genomics-guided application of clinically available XPO1 inhibitors.