<p>Pancorbo, Bruno Marco. Functional studies of ferritin 3-fold axis: effects of mutations near subunit interaction sites. (Under the direction of Dr. E. C. Theil)Ferritin is an iron storage protein whose metabolic importance is reflected in its ubiquitousness in living organisms. Ferritin is a multi-subunit protein (24 subunits) and one of its most interesting features is its 3-dimensional structure: a sphere-like structure with a 4-3-2 symmetry that has a hollow interior where iron is stored. This 3-dimensional structure is highly conserved among ferritins of different living organisms even when the homology of the primary structure of the different ferritins is as low as 22%. Such a degree of structural conservation can only be interpreted as the result of a near perfect balanced between ferritin's structure and function. Among the highly conserved residues are arginine 72 and aspartate 122 which form a salt bridge near the 3-fold interface. To study the importance of these residues in ferritin function, site-directed mutagenesis was used to disrupt and rescue this salt bridge. The properties of the mutants were tested and compared with those of the parent proteins.The greatest difference between mutants and parent proteins was seen in the amount of iron each released. Mutants were found to release a greater percentage of their initial iron than the parents released. Some mutants also showed an increased rate of iron release over the parent proteins, but the effect of the mutation differed depending on the type of subunits used. Another finding was that disruption of the salt bridge caused some of the ferritin subunits to have an increase in volume which seems to correlate with the difference in iron uptake rates for the different ferritin mutants.<P>

<p>Using traditional column chromatography, Protein DE has been purified from rat epididymides. Affinity, size exclusion, and ion-exchange chromatography were utilized to purify the protein to homogeneity. Protein DE purity was demonstrated using one and two-dimensional electrophoresis. Using the purified sample, an accurate molecular mass of 27,534 Daltons was determined using electrospray-ionization mass spectrometry. After four chromatographic steps, Protein DE was efficiently separated from all detectable epididymal proteins. This report provides the first rapid and reproducible method for purifying protein DE to homogeneity.Using western blot analysis and immunofluorescence, protein D is initially detected in rat epididymal tissue and associated with sperm from the distal caput region. In contrast, when sperm were recovered from the female reproductive tract seven hours after mating, protein D was not detected by western blot, but did display faint immunofluorescence. Additionally, using photoactivatible cross-linking, a 120 KD sperm membrane protein that specifically interacts with protein D was identified. A population of membrane bound protein D was released from NaCl washed epididymal sperm when incubated in the presence of phosphatidyl-inositol specific phospholipase C. This report is the first demonstrating that both the secretion and sperm-association of protein D occur in the distal caput region of the rat epididymis. It is the only report showing western blot analysis and immunolocalization of sperm-associated protein D on sperm deposited in the female reproductive tract after mating. Additionally, this is the first report that: (a) protein D binds specifically to a 120 KD membrane protein on the surface of epididymal sperm, (b) and that protein D is anchored or associated with a protein that is anchored to the sperm plasma membrane through a glycosylphosphatidyl inositol linkage<P>

Thesis (Ph.D.)--New York University, Graduate School of Arts and Science, 2006. / Typescript. Includes bibliographical references (leaves 112-127). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

Nodulin-intrinsic proteins (NIPs) are plant-specific, water and solute transporters with homology to soybean nodulin 26. In this study, it is shown that Arabidopsis NIP2;1 (AtNIP2;1) expression is acutely stimulated upon waterlogging (70-fold in whole seedlings within 1 hr) and hypoxia (> 1000-fold in roots within 2 hr). Subcellular localization of a AtNIP2;1::YFP fusion protein shows localization to the plasma membrane. Analysis of AtNIP2;1 protein in Xenopus oocytes shows that it is a transporter of lactic acid, a fermentation end product. Experiments with T-DNA insertional mutants in the AtNIP2;1 promoter showed that reduced AtNIP2;1 expression induced higher lactic acid accumulation in roots compared to wild type, both under normoxic and hypoxic conditions. Under normal growth conditions, atnip2;1 mutants grew normally but showed subtle changes in root morphology with increased numbers of lateral roots as well as increased primary root length and mass. Surprisingly, these T-DNA insertional mutants showed enhanced survival after severe hypoxia compared to wild type plants. Microarray analysis of a mutant (atnip2;1-1) and wild type roots showed that over 1300 transcripts were significantly upregulated in response to oxygen deprivation. Some genes were uniquely upregulated both under normoxia (54 genes) and hypoxia (14 genes) only in atnip2;1-1 roots. Overall, the data suggest that AtNIP2;1 is anaerobic-induced gene that encodes a lactic acid transporter, and may play a role in adaptation to lactic fermentation under anaerobic stress. Experiments with a second Arabidopsis NIP, AtNIP7;1 revealed specific expression in flowers,especially in developing pollen grains. A T-DNA insertional mutant (atnip7;1-1) showed no apparent defects in flower development under normal growth conditions. However, the mutant showed defects in pollen tube growth in the absence of boric acid, a known transport substrate for NIPs. Overall, these observations suggest that AtNIP7;1 might be involved in boric acid uptake necessary for pollen development in Arabidopsis.

Bioluminescence is a phenomenon in which chemical energy is converted into light energy. Here, the oxidation of a luciferin substrate, catalyzed by a luciferase enzyme, results in the emission of a photon. This biological process is exploited in a technology referred to as Bioluminescence Resonance Energy Transfer (BRET). As its name implies, BRET depends on a nonradiative energy transfer event that occurs between a donor luciferase and an acceptor fluorophore. Fusion of the donor and acceptor molecules to a protein(s) of-interest allows one to identify and monitor molecular events, such as protein interactions or hormone binding events, based solely on the spectral properties of the light produced. A primary goal of this research was to use BRET to investigate protein-protein interactions. Traditionally, BRET has been used to detect intermolecular interactions between protein pairs. To this end, BRET was applied to explore putative interactions between transcriptional regulators essential for organ polarity and floral development in Arabidopsis, FILAMENTOUS FLOWER (FIL), YABBY5 (YAB5), and LEUNIG (LUG). Results indicated that FIL and LUG are likely to interact in planta, supporting previous hypotheses that they function together within a protein complex. BRET has also been utilized to identify intramolecular, conformational changes that occur following a ligand-protein binding event. Hormone-binding sensors, in particular, have seen considerable success and are now used to monitor changes in small molecule concentrations within a cellular context. To identify whether BRET-based sensors are applicable to plant hormone studies, three sensors were created, incorporating AUXIN BINDING PROTEIN 1 (ABP1), GIBBERELLIC ACID INSENSITIVE DWARF 1A (GID1A), and CHICKEN THYROID HORMONE RECEPTOR, LIGAND BINDING DOMAIN (cTRá-1 LBD). Data obtained indicated that with modifications, single-molecule BRET sensors may be suitable for use in plants. Finally, luminescence imaging allows one to observe BRET as it occurs over a period of time. An imaging system was set up and used to monitor BRET occurring within transgenic Arabidopsis seedlings harboring either hRLUC-YFP or hRLUC constructs. Taken together, results from each of these studies highlight the versatility and sensitivity of BRET, and show that it can be used to monitor molecular events in a variety of applications.

Thesis (Ph. D.)--New York University, Graduate School of Arts and Science, 2005. / Typescript. Includes bibliographical references (leaves 133-156). Also available in electronic format on the World Wide Web. Access restricted to users affiliated with the licensed institutions.

This dissertation focuses on the biochemical and genetic characterization of the protein-protein interaction in the budding yeast Saccharomyces cerevisiae between the cytosolic translation initiation guanine nucleotide exchange factor eIF2B and the endoplasmic reticulum (ER) membrane-embedded very-long-chain fatty acid (VLCFA) synthesis beta-keto-reductase enzyme YBR159W (IFA38). The dissertation is divided between the physical characterization of the interaction and examination of the functional consequences the ybr159wΔ deletion has on the yeast cells physiology. I first look at how the interaction is occurring in yeast. I utilize yeast 2-hybrid analysis to show that eIF2B subunits GCD6 and GCD7 interact with YBR159W. My experiments show that eIF2B does not interact with other VLCFA synthesis enzymes and that YBR159W does not interact directly with the other canonical components of the eIF2B complex. Compared to a wild type strain, a ybr159wΔ null yeast strain has a reduced growth rate and the hallmarks of a reduced translation activity including reduced 35S-methionine incorporation and low levels of polyribosomes. It is unknown if the reduced translation rate is a direct or indirect consequence of the ybr159wΔ mutation. The total cellular abundance of eIF2B complex is reduced in a ybr159wΔ null strain but the stoichiometry of the eIF2B complex and its enzymatic activity appears equivalent to wild-type. Deletion of YBR159W or other VLCFA synthesis enzymes significantly alters sphingolipid production in yeast. Deletion of the eIF2B subunit GCN3 does not cause a significant change in sphingolipid production in yeast. In the second section, I examine what effect YBR159W has on the localization of the cytoplasmic eIF2B complex. In yeast, eIF2B forms one or two large foci known as eIF2B bodies. I discover that YBR159W is important for either the formation or maintenance of the eIF2B body. In ybr159wΔ null yeast, eIF2B forms many smaller foci throughout the cell. Other VLCFA synthesis enzyme mutants display this same phenotype. I also find that a fraction of the eIF2B complex associates with lipid membranes. This lipid association is not dependent on the presence of YBR159W and is not mediated by rough ER bound ribosomes. Further experiments are required to determine the mechanistic and functional role of YBR159W interacting with eIF2B.

PCSK9 is the third gene implicated in autosomal dominant hypercholesterolemia, due to its role in promoting the degradation of the low density lipoprotein receptor (LDLR). Little is known regarding the mechanism by which it promotes the degradation of LDLR, nor the effects PCSK9 has on other cellular proteins. I report here the first quantitative subcellular proteomic study of proteins affected by the expression of a variant of PCSK9. I show that the expression levels of 293 proteins were affected by the expression of the PCSK9-ACE2-V5 construct. Of particular interest, is a protein involved in receptor recycling, EHBP1, which shows reduced protein levels by both PCSK9-ACE2-V5 and the PCSK9-D374Y mutant. I show that an EHBP1 binding protein, EHD4, binds with PCSK9 and LDLR. These results establish novel effects of PCSK9 on liver cell protein levels, of which some relating to endosomal sorting are shown to bind to PCSK9 and LDLR in complex, providing insight into the mechanism of PCSK9 mediated LDLR degradation.

The Myeloid Translocation Gene (MTG) family was first discovered through the (8;21) translocation that leads to acute myeloid leukemia by fusing nearly all of Myeloid Translocation Gene 8 (MTG8) to an N-terminal portion of Acute Myeloid Leukemia 1 (AML1) and redirecting the normal function of MTG8 as a transcriptional co-repressor. The two other family members, Myeloid Translocation Gene 16 (MTG16) and Myeloid Tumor Gene Related-1 (MTGR1), are also implicated in leukemogenesis and function by forming corepressor complexes with proteins such as Nuclear Receptor Corepressor 1 (N-CoR) and histone deacetylases, recruiting them to transcription factor binding partners to regulate gene expression. To examine the physiological roles of Mtg16, we created a knock-out mouse model and found that deletion of Mtg16 perturbs hematopoietic stem cell function and affects both T and B-cell development, resulting in a reduced number of both developing thymocytes and mature B and T cells. Thorough characterization of the in vivo development of B and T cells found several changes throughout the development of both populations with the most significant changes in the stem, progenitor, and early lineage committed compartments. These changes are exacerbated after stress: both B and T cell development are nearly eliminated after bone marrow transplant and in vitro differentiation assays show striking deficits. While the defects to B and T cell development show similarity, mechanistic differences have become apparent. The defect in Mtg16(-/-) in vitro T cell development can be complimented with retroviral reintroduction of Mtg16 and we identified interactions with both the Notch Intracellular Domain and E2A as critical to the function of Mtg16 in specifying T-cell fate. The deficit in in vitro B-cell development in the absence of Mtg16 appears to be a growth deficit, and we have data to suggest that removal of p53 can overcome this deficit and restore in vitro growth. We hypothesize that through interactions with different transcription factors, Mtg16 regulates lymphoid lineage commitment, growth, and survival.

PCSK9 is the third gene implicated in autosomal dominant hypercholesterolemia, due to its role in promoting the degradation of the low density lipoprotein receptor (LDLR). Little is known regarding the mechanism by which it promotes the degradation of LDLR, nor the effects PCSK9 has on other cellular proteins. I report here the first quantitative subcellular proteomic study of proteins affected by the expression of a variant of PCSK9. I show that the expression levels of 293 proteins were affected by the expression of the PCSK9-ACE2-V5 construct. Of particular interest, is a protein involved in receptor recycling, EHBP1, which shows reduced protein levels by both PCSK9-ACE2-V5 and the PCSK9-D374Y mutant. I show that an EHBP1 binding protein, EHD4, binds with PCSK9 and LDLR. These results establish novel effects of PCSK9 on liver cell protein levels, of which some relating to endosomal sorting are shown to bind to PCSK9 and LDLR in complex, providing insight into the mechanism of PCSK9 mediated LDLR degradation.