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SINGLE-POINT MUTATION OF UBL4A ABOLISHES ITS INTERACTION WITH THE ARP2/3 COMPLEX
Title
SINGLE-POINT MUTATION OF UBL4A ABOLISHES ITS INTERACTION WITH THE ARP2/3 COMPLEX
Creator
Yao, Qi
Date
2018
Description
Ubl4A is a member of the ubiquitin-like protein family with multiple functions, such as pro-survival and anti-tumor. Our group previously...
Show moreUbl4A is a member of the ubiquitin-like protein family with multiple functions, such as pro-survival and anti-tumor. Our group previously found that Ubl4A directly interacts with actin-related protein Arp2/3 complex to promote Arp2/3-dependent actin “Y shape” branching formation. However, the binding region of Ubl4A for Arp2/3 still remains unknown. To address this question, we generated several Ubl4A mutant and truncated constructs, and cloned them into a GST vector in which GST was fused in-frame with Ubl4A at the N-terminus. We used Glutathione-beads to purify GST fusion proteins and performed pull-down assay with purified Arp2/3 complex. We show that C-terminus of Ubl4A is the region where the Arp2/3 complex interacts with. A single point mutation (D122A) in Ubl4A C-terminal “DYD” motif can abolish Ubl4A ability to bind Arp2/3. These results indicate that C-terminus, especially D122, is critical for Ubl4A association with Arp2/3.
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ENERGY METABOLISM OF CHLAMYDIA PNEUMONIAE
Title
ENERGY METABOLISM OF CHLAMYDIA PNEUMONIAE
Creator
McMillan, B. Julia
Date
2021
Description
Chlamydia pneumoniae is a gram-negative bacterium that infects the humanrespiratory tract. It causes acute pneumonia and has been linked to...
Show moreChlamydia pneumoniae is a gram-negative bacterium that infects the humanrespiratory tract. It causes acute pneumonia and has been linked to several chronic diseases including cardiovascular disease, asthma, and some neurological diseases. C. pneumoniae primarily exists in two forms, the elementary body (EB) and the reticulate body (RB). The EB infects host cells and the RB replicates inside them. In order to survive in and out of the host, it was thought that C. pneumoniae RBs obtain host ATP to use for energy, making it an “energy parasite.” However, genomic analysis indicated that it was also possible for C. pneumoniae to create ATP from its own respiratory chain using the Na + pump NADH Ubiquinone Oxidoreductase (Na + -NQR). Neither the details of the energy parasite theory nor the possibility of C. pneumoniae creating its own energy had been experimentally explored. This project used a pharmacological approach to explore C. pneumoniae host energy consumption at various developmental stages, examine a mechanism that the bacterium could use to produce its own energy, and assess the importance of a balanced Na + /H + gradient for energy production and maintaining homeostasis. Based on the genomic analysis, it was thought that C. pneumoniae would rely heavily on host ATP in the EB form but not the RB form, that inhibiting Na + -NQR would slow bacterial growth, particularly in RBs, and that disrupting the Na + /H + gradient would significantly reduce RB infection. The results indicate that in the EB form, C. pneumoniae relies on host ATP and requires a balanced Na + /H + gradient, but disrupting Na + -NQR does not hinder its growth. In the RB form, C. pneumoniae is not dependent on host ATP, nor on its own respiratory chain ATP, and is not impacted by an unbalanced Na + /H + gradient. Therefore, the energy parasite hypothesis appears to apply to C. pneumoniae EBs but not RBs. Furthermore, established C. pneumoniae infections are excellent at compensating for various environmental conditions and sources of energy, which proves challenging for drug design against C. pneumoniae.
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Evaluation of Bax∆2 Positive-Staining in Skin Samples Using Two Immunohistochemical Methods
Title
Evaluation of Bax∆2 Positive-Staining in Skin Samples Using Two Immunohistochemical Methods
Creator
Basheer, Sana
Date
2021
Description
BaxΔ2 is a pro-death and tumor suppressor protein that sensitizes cells to certain chemotherapies. Previous diaminobenzidine (DAB)-based...
Show moreBaxΔ2 is a pro-death and tumor suppressor protein that sensitizes cells to certain chemotherapies. Previous diaminobenzidine (DAB)-based staining revealed that Bax∆2 is found in all organs, including breast, colon, and skin tissues. In the skin, the Bax∆2 positive cells were mainly found in the basal cell layer of the epidermis with a few Bax∆2 positive cells in the connective tissue of the dermis, although their cellular identity was unknown. Previous literature has shown that melanin, which is found throughout the cells of the epidermis, is a brown color that provides no visual contrast to the DAB staining. While the DAB-based immunostaining showed cells that appeared to be Bax∆2 positive, this result needed to be confirmed. For this, a set of human skin samples from normal and cancerous tissue of various patients was examined. The co-staining of these samples for Bax∆2 and basal cells using immunofluorescence revealed that the apparent Bax∆2-positve DAB staining in epidermal basal cells and squamous cell carcinoma as false-positive, but the Bax∆2 positive cells found in the dermal connective tissue were not false positive—which is consistent with both previous DAB-based and fluorescence-based immunostaining. Using co-immunostaining for Bax∆2 with different cellular markers, the Bax2-positive cells in the connective tissue were identified potentially as macrophages and fibroblasts. Further studies are required to confirm the identity of the Bax∆2 positive cells in the connective tissue.
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ANALYTIC STUDY OF THE CELLULAR FUNCTIONS OF UBL4A
Title
ANALYTIC STUDY OF THE CELLULAR FUNCTIONS OF UBL4A
Creator
Zhang, Huaiyuan
Date
2021
Description
Ubiquitin-like protein 4A (Ubl4A) is a small protein encoded by a “housekeeping” gene that locates on the X chromosome. As a multi-functional...
Show moreUbiquitin-like protein 4A (Ubl4A) is a small protein encoded by a “housekeeping” gene that locates on the X chromosome. As a multi-functional protein, it has roles in a variety of cellular events including anti-tumorigenesis, response to DNA damage, inhibiting the fusion between autophagosome and lysosome, and docking of the tail-anchored proteins to the endoplasmic reticulum. We have previously reported that the newborns from Ubl4A-deficient mice had a high rate of mortality due to defect of AKT-dependent glucose metabolism. At the molecular level, Ubl4A directly binds with the actin related protein (Arp) 2/3 complex to accelerate the building up of the actin branching network, which further promotes the translocation and activation of the Akt, a key kinase for multiple cellular processes, from the cytosol to the plasma membrane.In further exploration of the molecular basis of Ubl4A in cell survival, here, we demonstrated that Ubl4A is critical for mitochondrial fusion and cell survival under nutrient depletion. In WT (wild-type) cells, the association of Ubl4A and the Arp2/3 complex serves as a primed “pool” of the actin branching network near mitochondria and enables mitochondria to fuse quickly for energy conservation upon starvation insult. However, such a “ready-to-go pool” of mitochondria was significantly decreased in the Ubl4A-deficient cells. As the result, the mitochondria became fragmentated, exhibited decreased trans-membrane potential, and accumulated ROS (reactive oxygen species), consequently, initiated mitochondria-mediated apoptosis. In this study, we also observed that Ubl4A-deficient mice displayed type II diabetic phenotype under a high-fat diet feeding. The preliminary results showed that these Ubl4A-deficient mice were more sensitive to glucose intolerance than their WT littermates, most likely owing to a delay in glucose uptake, and/or insulin secretion, both of which require the Arp2/3-actin branching network. We speculated that Ubl4A might be involved in cellular vesicle formation and/or secretion, but further investigation is needed to approve this hypothesis. Taken together, these findings provide a novel function of Ubl4A and further insight into the multi-functional roles of Ubl4A in mammalian cells, as well as the molecular basis for understanding the clinical relevance of Ubl4A in related human diseases.
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AMPLIFICATION AND PURIFICATION OF RECOMBINANT PRO-DEATH BAXΔ2 PROTEINS FOR STRUCTURE ANALYSIS
Title
AMPLIFICATION AND PURIFICATION OF RECOMBINANT PRO-DEATH BAXΔ2 PROTEINS FOR STRUCTURE ANALYSIS
Creator
Zhou, Yi
Date
2020
Description
BaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from...
Show moreBaxΔ2 is an isoform of the pro-apoptotic Bax family of proteins, which is an important anti-cancer protein. BaxΔ2 behaves differently from Baxα to induce apoptosis. The current computationally predicted model of BaxΔ2 is based on known Baxα structure, which is considered biased. Therefore, the elucidation of the BaxΔ2 crystal structure is critical. The goal of this project was to obtain a sufficient amount of purified recombinant Bax∆2 protein for crystallization. We cloned full-length BaxΔ2 fused with a poly-histidine tag on either N-terminus (His-Bax∆2) or C-terminus (Bax∆2-His) into an inducible bacterial expression vector. We found that His-Bax∆2 proteins were expressed better than Bax∆2-His, which totally inhibit host growth. However, the protein concentration of His-Bax∆2 was still too low to be detected by Coomassie blue staining. To increase His-Bax∆2 expression and avoid cytotoxicity, we further tested different bacterial host cells and applied the chaperone system. However, all attempts could not overcome Bax∆2 cytotoxicity and the protein expression levels were not high enough to be feasible for further large-scale purification. The mechanism underlying how Bax∆2 inhibits bacterial growth is still a mystery because Bax∆2 eukaryotic targets (mitochondria and caspases) do not exist in bacteria. Further experiments are required to explore the mechanism of Bax∆2 cytotoxicity in bacteria, so as to finally optimize and elevate the BaxΔ2 protein yields.
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