CHARACTERIZATION OF THE DYRK1A PROTEIN-PROTEIN INTERACTION NETWORK

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is a protein kinase encoded by a dosage-dependent gene. An extra copy of DYRK1A contributes to Down syndrome (DS) pathogenesis while loss of one allele causes severe mental retardation and autism. DYRK1A is involved in phosphorylation of several proteins that regulate cell cycle control and tumor suppression. However, the function and regulation of this kinase is not well understood and current knowledge does not fully explain dosage-dependent function of this important kinase. Our previous proteomic studies identified several novel DYRK1A interacting proteins including RNF169, FAM117B, TROAP, LZTS1, LZTS2 and DCAF7. In this dissertation, we report the proteomic, biochemical and functional characterization of this DYRK1A protein-protein interaction network. Firstly, we show that DYRK1A regulates that recruitment of 53BP1 to DNA double strand breaks (DSBs) in part through its interaction with RNF169. This revealed a novel role of DYRK1A in DSB repair pathway choice. Secondly, we identify LZTS1 and LZTS2 as novel regulators of DYRK1A activity towards LIN52. Thirdly, we observed that DCAF7 interacts with several other DYRK1A-binding proteins including RNF169, TROAP, FAM117B, LZTS1 and LZTS2 giving rise to various multi-subunit protein complexes, but it does not act as a scaffold for these interactions. We also observed an unexpected role of DYRK1A in mediating the interaction between these DYRK1A-binding proteins and DCAF7, which could explain some aspects of the dosage-dependent function of DYRK1A. As DCAF7 was the most highly enriched DYRK1A-interacting protein, we generated and analyzed the DCAF7 interactome in order to understand the functional significance of the DYRK1A-DCAF7 interaction. We show that DCAF7 interacts with the components of a multi-subunit Polycomb Repressive Complex 1.3/5 (PRC1.3/5) independent of DYRK1A, but DYRK1A could influence the molecular size of the PRC1.3/5 complex. Furthermore, our data suggest that DYRK1A and DCAF7 regulate the monoubiquitination of H2A at K119 by PRC1.3/5. Using RNA-seq analysis, we identified a common set of genes regulated by DYRK1A and DCAF7. Our data shows that DCAF7 requires DYRK1A for its transcriptional effect. Future studies will be focused to determine the molecular mechanism by which DYRK1A and DCAF7 regulate the transcription of the PRC1.5 target genes. In conclusion, DYRK1A interacting proteins could regulate the activity and function of DYRK1A and play a role in its biological functions including tumor suppression, the DNA damage response and transcription

Molecular Mechanisms of the DYRK1A-regulated DNA Repair

Molecular Mechanisms of the DYRK1A-regulated DNA Repair Polina Bukina, Dept. of Biology, with Dr. Sarah Golding, Dept. of Biology The functions of human Dual-specificity tyrosine (Y)-Regulated Kinase 1A, or DYRK1A, include cell cycle control and differentiation. DYRK1A is required for assembly of the DREAM complex and repression of the cell cycle-dependent genes, such as BRCA1 and RAD51, in quiescence. Our lab previously reported that overexpression of DYRK1A inhibits the accumulation of a DNA repair protein 53BP1, at the DNA double-stranded breaks (DSB). Accumulation of 53BP1 is attributed to repair by non-homologous end joining (NHEJ) over homologous recombination (HRR). The function of 53BP1 is opposed by RNF169, a ubiquitin-binding protein that also accumulates at the DSB sites and promotes HRR. It was found that DYRK1A interacts with RNF169 to regulate the displacement of 53BP1 from the DSB sites. This study focuses on RNF169 in order to understand the role of DYRK1A in DNA damage response. We used the Multi-Dimensional Protein Identification Technology (MudPIT) proteomic analysis to identify RNF169-interacting proteins. Human cancer U-2 OS cells stably expressing HA-tagged RNF169, as well as control cells were used for immunoprecipitation. The samples were sent to Stowers Institute for Medical Research for MudPIT proteomic analysis. In order to understand the regulation of DNA repair by DYRK1A, the RNA sequencing dataset was analyzed as part of other studies in the lab. The expression of the mRNA for repair factors RAD51 and BRCA1 was found to be regulated by DYRK1A. To determine the significance of this finding, an experiment was designed to assess BRCA1 and RAD51 protein levels in the normal U-2 OS cells and in the cells lacking DYRK1A (U-2 OS DYRK1A knockout cells) after inducing DNA damage by gamma irradiation. It was found that the levels of RAD51, BRCA1 and 53BP1 levels were increased with DYRK1A KO. These results were consistent with the finding that DNA repair efficiency is increased with DYRK1A KO. Further studies can help to understand if these effects are mediated by DYRK1A-regulated DREAM complex.https://scholarscompass.vcu.edu/uresposters/1357/thumbnail.jp

Zircon dissociation in air plasma through a low power transferred arc plasma torch

Thermal plasma dissociation offers a convenient and attractive route to prepare zirconium oxide from zircon mineral. Transferred and non-transferred arc plasma torches have been used to study zircon dissociation. The major thrust has been to accomplish complete dissociation and make the process simpler and cost effective. Technologically, this has been attempted in argon-fired plasma reactors using higher electrical power. The present work reports a cost effective low power transferred arc plasma (TAP) processing method for dissociating zircon by using air as the plasma forming gas. Phase composition and microstructure formation of the dissociated zircon were examined by XRD and SEM with EDX. Experimental results showed that the torch input power and processing time strongly influenced the dissociation percentage as well as the microstructure formation. Further, obtained results revealed that the air plasma medium significantly improved the percentage of zircon dissociation rather than argon plasma medium at 10 and 15 kW power levels. The air plasma gives complete zircon dissociation at 10 kW power with 5 min of processing

UNDERSTANDING THE FUNCTION OF DYRK1A THROUGH CHARACTERIZATION OF ITS INTERACTING PROTEINS

DYRK1A is a protein kinase encoded by a gene implicated in Down syndrome pathogenesis. Loss of DYRK1A could promote oncogenic transformation. However, the regulation and substrates of DYRK1A are not fully understood. MudPIT proteomic analysis revealed novel DYRK1A interacting proteins with poorly characterized or even unknown functions. Therefore, the aim of this thesis was to understand the function of DYRK1A through the characterization of its interacting proteins. To achieve this aim, we established stable cell lines expressing these proteins and confirmed the interactions between DYRK1A and seven candidate binding partners. Furthermore, we found that all novel DYRK1A-interacting proteins also bind DCAF7, a previously reported DYRK1A-binding scaffold protein that binds to the N-terminus of DYRK1A. Using cyto-nuclear fractionation and immunostaining we found that DYRK1A-interacting proteins were present in different cellular compartments, suggesting that DYRK1A could play distinct roles in the cell depending on its localization. DYRK1A has been shown to regulate cell proliferation and actin cytoskeleton therefore we used cell proliferation assays and actin staining to determine the role of DYRK1A-interacting proteins in these processes. Here we report functional characterization of the interacting partners of DYRK1A and present cell-based models that will help to understand the function and regulation of this important protein kinase

The cell cycle regulatory DREAM complex is disrupted by high expression of oncogenic B-Myb.

Overexpression of the oncogene MYBL2 (B-Myb) is associated with increased cell proliferation and serves as a marker of poor prognosis in cancer. However, the mechanism by which B-Myb alters the cell cycle is not fully understood. In proliferating cells, B-Myb interacts with the MuvB core complex including LIN9, LIN37, LIN52, RBBP4, and LIN54, forming the MMB (Myb-MuvB) complex, and promotes transcription of genes required for mitosis. Alternatively, the MuvB core interacts with Rb-like protein p130 and E2F4-DP1 to form the DREAM complex that mediates global repression of cell cycle genes in G0/G1, including a subset of MMB target genes. Here, we show that overexpression of B-Myb disrupts the DREAM complex in human cells, and this activity depends on the intact MuvB-binding domain in B-Myb. Furthermore, we found that B-Myb regulates the protein expression levels of the MuvB core subunit LIN52, a key adapter for assembly of both the DREAM and MMB complexes, by a mechanism that requires S28 phosphorylation site in LIN52. Given that high expression of B-Myb correlates with global loss of repression of DREAM target genes in breast and ovarian cancer, our findings offer mechanistic insights for aggressiveness of cancers with MYBL2 amplification, and establish the rationale for targeting B-Myb to restore cell cycle control

Bioactive and Tribological Behaviour of Atmospheric Plasma Sprayed Hydroxyapatite Coatings Reinforced by Lanthanum Oxide

Lanthanum oxide (La2O3) reinforced Hydroxyapatite coating was deposited by using unique gas tunnel type plasma spray torch under optimum spraying conditions. The phase and microstructure of the as-prepared powder and coatings were characterized by X-ray diffraction (XRD) and scanning electron microscope (SEM). In vitro bioactivity of the plasma sprayed lanthanum oxide reinforced hydroxyapatite coatings were investigated by using simulated body fluid solution. Results showed that there was onset of apatite formation on the surface of coatings after 15 days of immersion in SBF, while after 19 days of immersion in SBF it was indicated that a HCAp phase crystallized on their surface. Our studies demonstrate that lanthanum oxide reinforced hydroxyapatite coatings are potentially useful biomaterials with good tribological and bioactive behaviour

Optical Imaging With a High-Resolution Microendoscope to Identify Cholesteatoma of the Middle Ear

Objectives/Hypothesis: High-resolution optical imaging is an imaging modality that allows visualization of structural changes in epithelial tissue in real time. Our prior studies using contrast-enhanced microendoscopy to image squamous cell carcinoma in the head and neck demonstrated that the contrast agent, proflavine, has high affinity for keratinized tissue. Thus, high-resolution microendoscopy with proflavine provides a potential mechanism to identify ectopic keratin production, such as that associated with cholesteatoma formation, and distinguish between uninvolved mucosa and residual keratin at the time of surgery. Study Design: Ex vivo imaging of histopathologically confirmed samples of cholesteatoma and uninvolved middle ear epithelium. Methods: Seven separate specimens collected from patients who underwent surgical treatment for cholesteatoma were imaged ex vivo with the fiberoptic endoscope after surface staining with proflavine. Following imaging, the specimens were submitted for hematoxylin and eosin staining to allow histopathological correlation. Results: Cholesteatoma and surrounding middle ear epithelium have distinct imaging characteristics. Keratin-bearing areas of cholesteatoma lack nuclei and appear as confluent hyperfluorescence, whereas nuclei are easily visualized in specimens containing normal middle ear epithelium. Hyperfluorescence and loss of cellular detail is the imaging hallmark of keratin, allowing for discrimination of cholesteatoma from normal middle ear epithelium. Conclusions: This study demonstrates the feasibility of high-resolution optical imaging to discriminate cholesteatoma from uninvolved middle ear mucosa based on the unique staining properties of keratin. Use of real-time imaging may facilitate more complete extirpation of cholesteatoma by identifying areas of residual disease

Insights from the protein interaction Universe of the multifunctional “Goldilocks” kinase DYRK1A

Human Dual specificity tyrosine (Y)-Regulated Kinase 1A (DYRK1A) is encoded by a dosage-dependent gene located in the Down syndrome critical region of human chromosome 21. The known substrates of DYRK1A include proteins involved in transcription, cell cycle control, DNA repair and other processes. However, the function and regulation of this kinase is not fully understood, and the current knowledge does not fully explain the dosage-dependent function of this kinase. Several recent proteomic studies identified DYRK1A interacting proteins in several human cell lines. Interestingly, several of known protein substrates of DYRK1A were undetectable in these studies, likely due to a transient nature of the kinase-substrate interaction. It is possible that the stronger-binding DYRK1A interacting proteins, many of which are poorly characterized, are involved in regulatory functions by recruiting DYRK1A to the specific subcellular compartments or distinct signaling pathways. Better understanding of these DYRK1A-interacting proteins could help to decode the cellular processes regulated by this important protein kinase during embryonic development and in the adult organism. Here, we review the current knowledge of the biochemical and functional characterization of the DYRK1A protein-protein interaction network and discuss its involvement in human disease