Browsing by Author "Mayo, Lindsey"
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Item The Eukaryotic SMC5/6 Complex Represses the Replicative Program of High-Risk Human Papillomavirus(2020-10) Gibson, Ryan Taylor; Androphy, Elliot; Guo, Haitao; Yu, Andy; Mayo, LindseyHuman papillomaviruses (HPVs) are non-enveloped, circular double-stranded DNA viruses that infect basal keratinocytes of stratified squamous epithelia. High-risk HPV (HR-HPV) infection causes nearly all cervical cancers and an increasing number of head and neck cancers. While prophylactic vaccinations have reduced the incidence of HPV infection and attributable cancers, currently there is no cure for pre-existing HPV infection. As such, HPV remains a global health threat and a better understanding of HPV biology remains of significant medical importance for identification of novel therapeutic targets. The multi-subunit structural maintenance of chromosomes 5/6 complex (SMC5/6) is comprised of SMC5, SMC6 and NSE1-4. SMC5/6 is essential for homologous recombination DNA repair and reportedly functions as an antiviral factor during hepatitis B and herpes simplex-1 viral infections. Intriguingly, SMC5/6 has been found to associate with HR-HPV E2 proteins, which are multifunctional transcription factors essential to regulation of viral replication and transcription. The function of SMC5/6 associations with E2, as well as its role during HR-HPV infection remain unclear and we explored this question in the context of HR-HPV- 31. SMC6 interacted with HPV-31 E2 and co-immunoprecipitation of SMC6/E2 complexes required the E2 transactivation domain, inferring SMC6 association is limited to the full-length E2 isoform. Depletion of SMC6 and NSE3 increased HPV replication and transcription in keratinocytes stably maintaining episomal HPV-31, suggesting that the SMC5/6 complex represses these processes. Neither SMC6 nor NSE3 co-IP the viral E1 DNA helicase alone or E1/E2 complexes but the association of SMC6 with E2 was reduced in the presence of E1, indicating that SMC6 competes with E1 for E2 binding. This infers that SMC6 repression of the viral replicative program may involve inhibiting initiation of viral replication by disrupting E2 interactions with E1. Chromatin immunoprecipitation determined that SMC6 is present on episomal HPV-31 genomes, alluding to a possible role for SMC5/6 in modifying the chromatin state of viral DNA. Taken together, these findings describe a novel function for SMC5/6 as a repressor of the HPV-31 replicative program.Item HPV replication regulation by acetylation of a conserved lysine in the E2 protein(2017-06-26) Thomas, Yanique Serge Gillana; Androphy, Elliot J.; Klemsz, Michael; Yu, Andy; Mayo, Lindsey; Lee, Suk-HeePapillomaviruses (PVs) are non-enveloped DNA viruses that are the primary etiological agents of cervical and oropharyngeal cancers. Vaccines for H(human)PV have proven to be effective prophylactic treatments; however, there is no treatment available for those currently infected. To develop new therapies, we require a clear understanding of viral pathogenesis and regulation. The Papillomavirus E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in B(bovine)PV that is involved in the regulation of viral transcription. K111 is conserved in most papillomaviruses, so we pursued a mutational approach to query the functional significance of lysine in HPV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. When directly investigating origin unwinding, the replication defective E2 K111R mutant recruited E1 to the viral replication origin, but surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine K111 mutant increased origin melting and stimulated replication compared to wild type E2. We have identified Topoisomerase I as a key host factor involved in viral replication whose recruitment is dependent on K111 acetylation, and propose a new model for viral origin dynamics during replication initiation. This work reveals a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111.Item MiR-10a as a Modulator of Proliferation and Cell Cycle Progression in Ovarian Clear Cell Carcinoma(2024-08) Collins, Kaitlyn Elizabeth; Hawkins, Shannon; Kim, Jaeyeon; Mayo, Lindsey; Nephew, Kenneth; Zhang, Chi; Zimmers, TeresaEndometriosis, a benign inflammatory disease whereby endometrial-like tissue grows outside the uterus, is a significant risk factor for endometriosis-associated ovarian cancers. In particular, ovarian endometriomas, cystic lesions of deeply invasive endometriosis, are a potential precursor lesion for ovarian clear cell carcinoma (OCCC). To explore the transcriptomic landscape, OCCC from women with pathology-proven concurrent endometriosis (n=4) were compared to benign endometriomas (n=4) by bulk RNA and small-RNA sequencing. Analysis of protein-coding genes identified 2449 upregulated and 3131 downregulated protein-coding genes (DESeq2, P<0.05, log2 fold-change>|1|) in OCCC with concurrent endometriosis compared to endometriomas. Gene set enrichment analysis showed upregulation of cell cycle regulation and DNA replication pathways and downregulation in cytokine receptor signaling and matrisome pathways. Analysis of miRNAs revealed 64 upregulated and 61 downregulated mature miRNA molecules (DESeq2, P<0.05, log2 fold-change>|1|). Hsa-miR-10a-5p represented over 21% of the miRNA molecules in OCCC with endometriosis and was significantly upregulated (NGS: log2 fold change=4.37, P=2.43E-18; QPCR: 8.1-fold change, P<0.05). Correlation between miR-10a expression level in OCCC cell lines and IC50 (50% inhibitory concentration) of carboplatin in vitro revealed a positive correlation (R2=0.92). The cellular function of miR-10a was investigated by overexpressing miR-10a in vitro. MiR-10a overexpression revealed a significant decrease in proliferation (n=6; P< 0.05), compared to a non-targeting control. Cell-cycle analysis revealed a significant shift in cells from S and G2 to G1 in (n=6; P<0.0001). MiR-10a overexpression in vitro was correlated with decreased expression of predicted miR-10a target genes critical for proliferation, cell-cycle regulation, and cell survival [SERPINE1 (3.2 downregulated; P<0.05), CDK6 (2.4 downregulated; P<0.05) and, RAP2A (2-3 downregulated; P<0.05)].Item Nuclear PTEN enhances the maturation of a microRNA regulon to limit MyD88-dependent susceptibility to sepsis(American Association for the Advancement of Science, 2018-05-01) Sisti, Flavia; Wang, Soujuan; Brandt, Stephanie L.; Glosson-Byers, Nicole; Mayo, Lindsey; Son, Young min; Sturgeon, Sarah; Filgueiras, Luciano; Jancar, Sonia; Wong, Hector; Dela Cruz, Charles S.; Andrews, Nathaniel; Alves-Filho, Jose Carlos; Cunha, Fernando Q.; Serezani, C. Henrique; Microbiology and Immunology, School of MedicineSepsis-induced organ damage is caused by systemic inflammatory response syndrome (SIRS), which results in substantial comorbidities. Therefore, it is of medical importance to identify molecular brakes that can be exploited to dampen inflammation and prevent the development of SIRS. We investigated the role of phosphatase and tensin homolog (PTEN) in suppressing SIRS, increasing microbial clearance, and preventing lung damage. Septic patients and mice with sepsis exhibited increased PTEN expression in leukocytes. Myeloid-specific Pten deletion in an animal model of sepsis increased bacterial loads and cytokine production, which depended on enhanced myeloid differentiation primary response gene 88 (MyD88) abundance and resulted in mortality. PTEN-mediated induction of the microRNAs (miRNAs) miR125b and miR203b reduced the abundance of MyD88. Loss- and gain-of-function assays demonstrated that PTEN induced miRNA production by associating with and facilitating the nuclear localization of Drosha-Dgcr8, part of the miRNA-processing complex. Reconstitution of PTEN-deficient mouse embryonic fibroblasts with a mutant form of PTEN that does not localize to the nucleus resulted in retention of Drosha-Dgcr8 in the cytoplasm and impaired production of mature miRNAs. Thus, we identified a regulatory pathway involving nuclear PTEN-mediated miRNA generation that limits the production of MyD88 and thereby limits sepsis-associated mortality.Item Regulation of papillomavirus E2 protein by posttranslational modification(2015-04-24) Culleton, Sara Poirier; Androphy, Elliot J.; Klemsz, Michael; Mayo, Lindsey; Nakshatri, Harikrishna; Sullivan, William J., Jr.Papillomaviruses (PVs) are small, double-stranded DNA viruses. Hundreds of species have evolved to replicate in mammals, birds, and reptiles. Approximately two hundred species are estimated to infect humans alone, and these human papillomaviruses (HPVs) cause diseases ranging from benign warts to anogenital and oropharyngeal cancers. While vaccination is effective at preventing the majority of these infections and their disease outcomes, there are no successful treatments for existing infections; thus, exploration of novel therapeutic targets is warranted. PVs control expression and function of their gene products through alternative splicing, alternate start codons, and post-translational modification (PTM). The viral E2 protein regulates transcription, replication, and genome maintenance in infected cells, and PTMs have been demonstrated for E2 proteins from multiple papillomavirus types. Serine phosphorylation events were reported to influence E2 stability, and our laboratory was the first to describe in vitro acetylation events with implications for E2 transcription function. Here we report confirmation of these acetylation events in vivo and additional data elucidating the role of these PTMs in viral transcription. Moreover, we present a novel phosphorylation site for bovine papillomavirus type 1 (BPV-1) E2 at tyrosine 102 (Y102). Using phospho-deficient and phospho-mimetic point mutants, we found that this site influences E2-mediated transcription and replication, and we hypothesize that phosphorylation at Y102 regulates these activities by interrupting the association of E2 with its binding partners. We also report interaction of BPV-1 E2 and HPV-31 E2 with different receptor tyrosine kinases (TKs), most notably members of the fibroblast growth factor receptor family. We hypothesize that Y102 phosphorylation by these receptors occurs early in infection to limit viral replication and gene expression. Further studies will cement the role of RTKs in PV biology and could reveal novel therapeutic strategies.Item Restriction of Glioma Progression and Mesenchymal Characteristics by Angiomotin-like 1(2020-10) Lange, Kevin Clayton; Wells, Clark D.; Dong, X. Charlie; Ivan, Mircea; Mayo, Lindsey; Wek, RonaldAngiomotin-like 1 (AmotL1) serves as a scaffold for protein complexes that promote cell polarity and HIPPO signaling to enable their suppression of oncogenic phenotypes in multiple epithelial-derived cancers. In this study, an analysis of multiple tumor databases revealed that AmotL1 transcript levels associate with positive survival and reduced tumor grade in astrocytomas. The suppression of AmotL1 transcript levels was most prevalent in in glioblastoma tumor regions that are associated with invasion and mesenchymal-like transcriptional profiles. Factors associated with tumor progression were consequently causally linked to AmotL1 expression in normal astrocytes and glioblastoma cells. While most tumor suppressive effects of AmotL1 are related to its regulation of YAP and TAZ, the potent effects of AmotL1 down-regulation were found to be independent of these two oncoproteins. Further, AmotL1 was shown to inhibit Wnt signaling through binding of the Fzd4 receptor via MAGI-3. Such binding was associated with an ability by AmotL1 to redistribute Fzd4 from the cell surface to intracellular complexes with AmotL1 and MAGI-3. AmotL1 was also shown to be transcriptionally suppressed under hypoxia by HIF2α. This suppression was found to promote invasion by increasing levels of c-MET. These results show that hypoxia suppresses AmotL1 to promote a likely mesenchymal transition. These effects help to explain the association of AmotL1 down-regulation in glioblastomas with increased tumor grade and poor patient survival.Item Specific Functions of the Tumor Suppressor P53 are Activated by P73 and VHL(2019-07) Wolf, Eric R.; Mayo, Lindsey; Goebl, Mark; Ivan, Mircea; Mendonca, Marc; Wells, ClarkThe transcription factor and tumor suppressor protein p53 critically regulates cell survival or death in response to cellular stress. p53 can activate genes involved in a wide variety of processes, including apoptosis, cell cycle arrest, angiogenesis, metabolism, and senescence. Mutations in p53 are common in cancer and alter its interactions with other proteins, but there are other mechanisms and posttranslational modifications that can alter these interactions as well. In some tumors, such as renal cell carcinoma, p53 is commonly inactive even though mutations to TP53 are rare. This suggests that there are other biochemical mechanisms of inhibition, which we explore in this study. Mutations in the DNA-binding domain of p53 result in conformational changes that enable p53 to interact with and inhibit its family member p73, thereby promoting cell survival instead of apoptosis. In contrast, it has been reported that wild-type p53 does not bind to p73. We found that JNK-mediated phosphorylation of Thr81 in the proline-rich domain (PRD) of p53 enabled wild-type p53 to form a complex with p73. The dimerization of wild-type p53 with p73 facilitated the expression of apoptotic target genes such as PUMA and BAX, as well as the induction of apoptosis. In addition to the apoptotic function of p53, the tumor suppressor also plays a major role in the inhibition of angiogenesis. Here we also report a new mechanism where the Mdm2 oncoprotein can indirectly inactive p53 through the regulation of the tumor suppressor VHL. In response to hypoxia, VHL can bind p53, which results in activation of several anti-angiogenic targets of p53 such as THBS1 and COL18A1. Mdm2 regulates the VHL-p53 interaction by conjugating nedd8 to VHL within a region that is important for the VHL-p53 interaction, blocking the induction of anti-angiogenic genes and resulting in a proangiogenic phenotype. Due to its positive regulation of major proangiogenic proteins and its negative regulation of potent inhibitors of angiogenesis, we propose that the oncoprotein Mdm2 is the angiogenic switch. These findings refine our understanding of p53 interactions and activation, specifically for p53-p73 induced cell death and p53-VHL inhibition of angiogenesis.Item STAINING OF OVCA1 ANTIBODY IN HUMAN MALIGNANCIES(Office of the Vice Chancellor for Research, 2012-04-13) Grothaus, Kristen; Temm, Connie; Mayo, Lindsey; Sandusky, GeorgeImmunohistochemistry biomarkers are currently being developed to tar-get specific proteins found in cancer cells. The biomarker and putative tumor suppressor, OvCa1, has a function that is not well characterized. Due to lack of reagents, we developed monoclonal antibodies of OvCa1 to examine mul-tiple human malignancies. Primary cancers with different histologic grades as well as with metastatic lesions were examined with the monoclonal anti-bodies. Ovarian cancer tissue samples from the IU Simon Cancer Center Tis-sue Bank were used for this study. The samples were fixed in neutral buff-ered formalin and processed into a paraffin block. The slides were microtomed, and immunohistochemistry (IHC) with the OvCa1 antibody was performed. Thirty-one low, medium, and high grade tumors as well as meta-static ovarian carcinomas were evaluated. All cases revealed a range of staining intensity with OvCa1. The results indicated that OvCa1 had the highest immunostaining in the high grade, Stage 3 to 4 ovarian carcinomas. Medium grade tumors had less OvCa1 expression, while the metastatic tu-mors had less staining than any of the other three grades. Immunostaining was observed primarily in the cytoplasm and nucleus of the tumor cells. In addition, we evaluated approximately 20 tumors from various different or-gans. These included prostate, breast, spleen, lung, colon, stomach, and kidney tumors, which were positive for immunostaining with the OvCa1 anti-body. In summary, the results indicate that all histologic grades express the biomarker, OvCa1, and the staining intensity was highest in the high grade, Stage 3 and 4 tumors. Our preliminary studies demonstrate a further need to delineate OvCa1 as a potential biomarker, which could be used for early detection and diagnosis of ovarian cancer.