Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secreted glycoprotein that promotes degradation of low-density lipoprotein receptors. Gain- and loss-of-function variants of PCSK9 cause hypercholesterolemia and hypocholesterolemia, respectively. Although it has been a decade since the discovery of PCSK9, its effect in terms of global protein changes and interactions still require further understanding. This study provided a global outlook at the protein changes caused by PCSK9 and its variants in human hepatic HUH7 cell line. First, a proteomics-based method for protein subcellular distribution analysis has been developed. Second, through secretome analyses, six apolipoproteins and six proteins involved in the coagulation pathway were found with >2-fold changes between wild type PCSK9 and its variants. Third, through secreted interactome analyses, a list of 159 PCSK9 interactor candidates was identified. Two interacting proteins, FASN and PSMD2, were validated and demonstrated with dynamic interacting patterns between PCSK9 and its variants.

The first report of polyphosphates (polyP) was in 1890 by L. Liberman and since then, polyP’s role in biology has been explored. PolyPs are chains of phosphoanhydride-linked inorganic phosphates ranging from 3-1000s of units in length. These chains are implicated in many cellular pathways including blood clotting, bacterial virulence, and neuroproteotoxic disease. Given the diversity of polyP, they make an excellent candidate in the development of novel therapeutics. In yeast, polyP is synthesized by the vacuolar transporter chaperone (VTC) complex as a translocation event into the vacuole lumen. In 2015, polyP chains were found to act as a post-translational modification termed polyphosphorylation on yeast proteins (Nsr1 and Top1). This modification occurs non-enzymatically on lysine residues within poly-acidic, serine, and lysine (PASK) motifs and can only be detected via electrophoretic mobility shift on NuPAGE gels. We have since expanded the pool of yeast polyphosphorylated substrates to 25, with an enrichment of proteins with roles related to RNA biology. Additionally, we were the first group to demonstrate polyphosphorylation of 6 human proteins by expressing E. coli PPK1 in HEK293T cells. We next focused on elaborating how polyP is being regulated via the VTC complex by assessing which protein trafficking pathways are critical for VTC localization at the vacuole membrane. We found the adaptor protein 3 (AP-3) complex is responsible for localizing Vtc5 subunit to the vacuole membrane and in AP-3 mutants, Vtc5 becomes mislocalized to the vacuole lumen and degraded. Vtc5 degradation, upon AP-3 mutation, is mediated by the endosomal sorting complex required for transport (ESCRT) complex. The loss of polyP in AP-3 mutants is imparted by Vtc5 mislocalization. In humans, mutations in AP-3 cause a rare genetic disorder termed Hermansky-Pudlak Syndrome (HPS) which has a wide range of symptoms. These include defects in polyP accumulation in platelets, likely related to a loss of polyP. We expect that our work using yeast will provide a framework for understanding fundamental aspects of polyP biology related to HPS and other health conditions.

<p>Human testis expressed 261 (TEX261) protein is predicted to be involved in essential cellular pathways such as proliferation, apoptosis, and COPII-mediated intracellular trafficking, yet has been scarcely researched in human cell models. Since TEX261 dysregulation has been observed in HCC, investigating the role of TEX261 in hepatocytes is essential. In this study we utilized molecular cloning and fluorescent protein tags to visualize the expression of TEX261 and associated proteins SAR1A and ALPL by confocal microscopy. We observed that TEX261 is closely associated with both proteins, indicating that TEX261 may be involved in ALPL packing into the COPII complex responsible for intracellular trafficking from the ER to the Golgi apparatus. We assessed TEX261 role in apoptosis by measuring caspase 3 activity and observed that TEX261 overexpression induced apoptosis at a similar rate as the positive control but did not significantly increase apoptosis compared to the negative control. We also recorded cell proliferation by overexpressing and silencing TEX261 in two cell lines. Our data showed that altered TEX261 expression did not impact Thle-2 proliferation, but TEX261 overexpression did significantly decrease proliferation in the HCC cell line Hep3B2.1-7. Overall, our results suggest that TEX261 does play a role in intracellular trafficking, apoptosis and proliferation, yet future studies need to be done to further define its role in cell regulatory mechanisms. Better control of the experimental error seems to be required to define the function of TEX261 in apoptosis.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / At the present time, gentamicin is used in the treatment of both Gram-negative and Gram-positive bacterial infections. However, the poorly understood side effect of nephrotoxicity is a serious problem and is one of the dose-limiting factors in the use of gentamicin. In our model system, Saccharomyces cerevisiae, which is relatively resistant to gentamicin, at least 20 genes are required for gentamicin resistance. Inspection of the physical and genetic interactions of the gentamicin sensitive mutants reveals a network centered on the ARF pathway which plays a key role in the regulation of retrograde trafficking. Our studies show that arf1ts arf1Δ arf2Δ cells, gea1ts gea1Δ gea2Δ cells, and gcs1ts gcs1Δ glo3Δ cells are all hypersensitive to gentamicin which indicates that impaired Arf1 function causes yeast cells to become hypersensitive to gentamicin. As evidence, cellular CPY trafficking and processing are blocked by the presence of gentamicin in some of these mutants. Interestingly, gentamicin can directly affect the level of the GTP-bound form of Arf1 in a cell growth phase-dependent manner; even though total Arf1 levels in S. cerevisiae are not affected. As predicted, we also find that gentamicin-bound resin can enrich both yeast Arf1-TAP protein and rat Arf1 protein in vitro. With the help of mass spectrometry, we also generated a gentamicin-binding protein list. Gentamicin hypersensitivity is also observed in S. cerevisiae double deletion strains that lack both ARF1 and ARF2 but are kept alive by the presence of hARF4 or bARF1. Increased -1 programmed ribosomal frameshifting efficiency is also observed in cells treated with gentamicin. Finally, a comparison of a gentamicin mixture and four of the gentamicin congeners reveals that gentamicin C1 is less toxic than other gentamicin congeners or the gentamicin total mixture.

Despite intensive research, the cellular and molecular mechanisms involved in human cellular susceptibility to prion infection remain poorly defined, in part due to the continuing lack of cultured human cells that are susceptible to infection with human prions. Such culture models would present distinct advantages including speed and expense compared with animal models, and would provide systems in which to investigate the interaction between PrPC and PrPSc, the basis of cellular susceptibility, the nature of the species barrier and the mechanism of prion propagation in situ. This study sought to examine whether non-neuronal cells might provide opportunities to establish human cell lines replicating human prions. A human follicular dendritic cell-like cell line (termed HK) was obtained, further characterised and then tested for its ability to support human prion replication. The mechanisms of internalisation, intracellular trafficking and the eventual fate of exogenous PrPSc taken up by these cells were also examined. This thesis similarly examined the cellular response of human embryonic stem cells (hESC) to acute exposure to human and animal prions. PrPC was found to be abundantly expressed by HK cells and HK cell extracts were found to support conversion to PrPSc in a cell-free conversion assay. However, HK cells exposed to infectious brain homogenates failed to accumulate PrPSc or become infected in vitro. Exposed HK and hESC did display a readily detectable, time dependent uptake of PrPSc from medium spiked with prion-infected brain homogenates that was independent of the species, disease phenotype and PRNP codon 129 genotype of the human source and the recipient cells. The exposed cells showed intensely labelled intracellular accumulations of PrPSc with coarse granular morphology, largely in the juxtanuclear region of cytoplasm. However, when the brain-spiked medium was withdrawn and cells were given control medium, the intensity and extent of PrPSc immunostaining rapidly diminished. Co-localisation studies implicated caveolae-mediated endocytic uptake of exogenous PrPSc, apparently preceding uptake via clathrin coated pits in HK cells. Evidence suggesting that the endosomal recycling compartment and lysosomes are involved in intracellular trafficking and degradation of exogenous PrPSc was also found. Understanding the cell biology of these processes may help to explain why the majority of cultured cells are refractory to prion infection in vitro. Internalization of misfolded PrP and its subsequent degradation in the lysosomal compartment might function as a self-protective cellular mechanism, serving to eliminate non-native, presumably dysfunctional and potentially dangerous PrP conformers, whether generated endogenously or acquired through exposure to exogenous prion infectivity.

The focus of this investigation is about DNA topoisomerases, the molecular targets of clinically important chemotherapy, and mechanisms of drug resistance in human myeloma and leukemia cell lines. The ultimate goal of this investigation was to identify mechanism(s) of drug resistance to anticancer agents so that a strategy to overcome drug resistance could be conceived. We established an in vitro cell model by using human leukemia and myeloma cell lines to investigate possible mechanisms of drug resistance that are observed in confluent cells. Plateau cell densities demonstrated de novo drug resistance to commonly used chemotherapeutic agents that was independent of altered drug transport. We established that cellular drug resistance in these cells is a function of topo IIalpha subcellular localization and further demonstrate that topo IIalpha translocates to the cytoplasm in a cell-density dependent manner. We provide experimental data that supports the nuclear export of topo IIalpha as the most likely event contributing to drug resistance to topoisomerase II inhibitors, which occurs when transformed cells transition from log to plateau cell density. We provided a plausible nuclear export pathway for topo IIalpha, by identifying two Leptomycin B sensitive nuclear export signals, which are homologous to the binding sites recognized by the nuclear export receptor, exportin-1. Thus, topo IIalpha is likely to be exported from the nucleus at plateau cell densities when exportin-1 binds topo IIalpha. We confirmed that the nuclear export signals identified in topo IIalpha are functional when expressed in human myeloma cells transfected with an epitope-tagged topo IIalpha gene. Furthermore we demonstrate that the nuclear export signals can be abolished by site-directed mutagenesis of specific amino acids residues found in the nuclear export signal. Our data may have clinical relevance because plasma cells obtained from bone marrow aspirates of patients with multiple myeloma contain a cytoplasmic distribution of topo IIalpha. The potential implications of a functioning nuclear enzyme located in the cytoplasm of cells and theoretical mechanisms for overcoming the observed drug resistance are considered.

The establishment and maintenance of cell polarity during development is an active process that requires specific protein sorting and targeting to apical and basolateral regions of the cell. Our lab has identified an apical early endosomal marker, endotubin, in developing rat intestine, which we have used to label specialized apical endosomal tubules, and to probe for components of the apical sorting machinery. Studies with endotubin have implicated the small GTPase Rab14 as part of the sorting machinery for apical targeting. The current work pursues further study of the interaction between Rab14 and endotubin, as well as the role for Rab14 in the establishment of cell asymmetry. Interestingly, even nonpolarized cells may utilize polarized trafficking components for proper sorting and dynamics of endotubin.

xvii, 167 p. : ill. (some col.) / The vacuolar proton-translocating ATPase (V-ATPase) is a multisubunit enzyme complex responsible for acidification of cellular organelles. The V-ATPase hydrolyzes ATP to pump protons across membranes to create an electrochemical gradient. Acidification of vesicular compartments is critical in numerous biological processes including protein trafficking, endocytosis, and ion homeostasis; defects in V-ATPase function can also lead to human diseases. While the function of the V-ATPase enzyme is highly conserved across eukaryotes, the molecular architecture of this protein complex has undergone unique structural changes through evolutionary time. The goal of this work is to investigate the assembly, transport, and evolution of this critical molecular machine in the model organism <italic>Saccharomyces cerevisiae</italic>. A series of genetic screens was performed in budding yeast to identify factors and pathways that are involved in promoting full V-ATPase function. I utilized several "assembly factor" alleles to serve as sensitized genetic backgrounds to partially reduce enzyme function; this work implicated sphingolipid composition in promoting full vacuolar ATPase enzyme function. I also used ancestral gene reconstruction to analyze the two isoforms of subunit a of the V₀ subdomain (Vph1p and Stv1p) by recreating the most recent common ancestral subunit (Anc.a). Characterization of Anc.a demonstrated that this ancient subunit was able to properly assemble and function within a hybrid V-ATPase complex. While the Vph1p-containing complex localized to the vacuole membrane and the Stv1p-containing complex was present on the Golgi/endosome, incorporation of Anc.a caused the V-ATPase to localize to both types of cellular compartments. Finally, I used ancestral reconstruction to investigate the lineage-specific gene duplication of one of the proteolipid subunits of the V₀ subcomplex that occurred within the fungal clade. I demonstrate that inclusion of a third proteolipid subunit within fungi (as compared to two subunits within metazoans) could have occurred via neutral processes by asymmetric degeneration of subunit-subunit interfaces that "ratcheted" the duplicated subunit with the V₀ ring. These results present a model that describes how macromolecular machines can increase in complexity through evolutionary time. This dissertation includes previously published co-authored material and unpublished co-authored material. / Committee in charge: George Sprague, Chairperson; Tom H. Stevens, Advisor; Victoria Herman, Member; Bruce Bowerman, Member; Ken Prehoda, Outside Member

Protein quality control is crucial to maintaining cellular function. A failure to clear misfolded, aggregation prone proteins can lead to the accumulation of toxic protein aggregates that interfere with cellular pathways and lead to cell death. In addition, the degradation of partially functional proteins can lead to loss of function diseases. Understanding proteins quality control mechanisms is therefore of fundamental importance to understanding these disease pathways. Systems that operate to monitor the structure of soluble protein domains are now relatively well understood. However, in addition to soluble domains, membrane proteins contain regions that span lipid bilayers, and a key question that remains is where and how these transmembrane domains (TMDs) that fail to assemble correctly or are otherwise aberrant are recognised within subcellular compartments. As such, in this study model chimeric proteins containing the luminal and cytoplasmic domain of the single-spanning membrane protein CD8 and exogenous TMDs derived from polytopic membrane proteins were used to investigate the handling of non-native TMDs in the secretory pathway. CD8 chimeras containing non-native TMDs were found to be recognised by endoplasmic reticulum (ER) quality control pathways. Importantly, ER-associated degradation of CD8 chimeras containing exogenous TMDs was reliant upon ubiquitination of cytoplasmic lysine residues prior to retrotranslocation and dislocation from the ER membrane. In contrast, CD8 containing the endogenous TMD but a misfolded luminal domain could be efficiently degraded when cytoplasmic lysines were removed, suggesting that the retrotranslocation mechanisms for these proteins are distinct and defined by the domain which is misfolded. A proportion of the CD8 chimeras containing non-native TMDs were able to exit the ER, and were retrieved to the ER from the Golgi. Golgi-to-ER retrieval was found to be at least partially mediated by Rer1. CD8 chimeras that escaped ER retrieval could also be retained in the Golgi and subsequently degraded in lysosomes, indicating the presence of an as yet undefined TMD-based Golgi quality control checkpoint in mammalian cells. Furthermore, in contrast to WT CD8 which was stable at the plasma membrane, CD8 chimeras containing non-native TMDs that trafficked to the cell surface were rapidly internalised and sorted to lysosomes. This process was largely independent of the cytoplasmic domain of CD8, suggesting signals within the TMD induced internalisation of these CD8 chimeras. The proportion of the CD8 chimeras that trafficked to the plasma membrane, and the stability of the protein at the cell surface, was dependent upon the presence of polar residues within the TMDs, indicating that exposed polar residues in non-native TMDs may alter the handling of proteins at the Golgi and cell surface. Together, these results further our understanding of the mechanisms by which proteins containing aberrant transmembrane domains are handled at multiple subcellular compartments.

Nef, an accessory protein of HIV-1, is a critical determinant of viral pathogenicity. The pathogenic effects of Nef are in large part dependent on its ability to decrease the amount of CD4 on the surface of infected cells. Early studies suggested that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein-trafficking machinery. However, the specific sorting pathway that Nef uses to modulate CD4 expression remained uncertain. According to one model, Nef was thought to interfere with the transport of newly synthesized CD4 from the TGN to the cell-surface. Another model claimed that Nef facilitated the removal of CD4 from the plasma membrane. The primary goal of this thesis was to determine which of these models was correct. To accomplish this objective, a novel Nef-CD4 system was developed in Drosophila S2 cells. Nef was not only able to downregulate human CD4 in S2 cells, but it did so in a manner that was phenotypically indistinguishable from its activity in human cells. An RNAi screen targeting protein-trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP-2 complex in the Nef-mediated downregulation of CD4. In contrast, depletion of the related AP-1 and AP-3 complexes, which direct transport from the TGN and endosomes, had no effect. The requirement for AP-2 was subsequently confirmed in a human cell line. Yeast three-hybrid and GST pull-down assays were then used to demonstrate a robust, direct interaction between Nef and AP-2. This interaction was found to depend on a [D/E]xxxL[L/I]-type dileucine motif, located in the C-terminal loop of Nef, that is essential for CD4 downregulation. While mapping the binding site of AP-2 on Nef, a second determinant of interaction in the C-terminal loop was identified. Mutation of this motif, which conforms to a consensus [D/E]D diacidic sequence, prevented Nef from binding to AP-2 and down-regulating CD4. However, the same mutations did not affect the ability of Nef to interact with either AP-1 or AP-3, providing further evidence that these complexes are not required for the modulation of CD4 expression. Additional experiments indicated that the Nef diacidic motif most likely binds to a basic patch on AP-2 α-adaptin that is not present in the homologous AP-1 γ and AP-3 δ subunits. As with the Nef diluecine and diacidic motifs, the α-adaptin basic patch was shown to be necessary for CD4 downregulation. Moreover, all three of these motifs were needed for the cooperative assembly of a CD4-Nef-AP-2 tripartite complex, which was observed here for the first time using a yeast four-hybrid system. The data in this thesis uniformly support an endocytic model of Nef-mediated CD4 downregulation. Indeed, there is now strong evidence that Nef simultaneously binds CD4 and AP-2, thereby connecting the receptor to the cellular endocytic machinery and promoting its rapid internalization from the plasma membrane. In addition, the identification of novel motifs required for this process has provided new insights on endocytosis, and may facilitate the development of pharmacological inhibitors of Nef function.