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Indiana University-Purdue University Indianapolis (IUPUI) / The  cardiac  voltage-­gated  sodium  channels  (Nav1.5)  play  a  specific  and   critical  role  in  regulating  cardiac  electrical  activity  by  initiating  and  propagating   action  potentials  in  the  heart.  The  association  between  Nav1.5  dysfunctions  and   generation  of  various  types  of  cardiac  arrhythmia  disease,  including  long-­QT3   and  Brugada  syndrome,  is  well  established.  Many  types  of  post-­translational   modifications  have  been  shown  to  regulate  Nav1.5  biophysical  properties,   including  phosphorylation,  glycosylation  and  ubiquitination.  However,  our   understanding  about  how  post-­translational  lipid  modification  affects  sodium   channel  function  and  cellular  excitability,  is  still  lacking.  The  goal  of  this   dissertation  is  to  characterize  Nav1.5  palmitoylation,  one  of  the  most  common   post-­translational  lipid  modification  and  its  role  in  regulating  Nav1.5  function  and   cardiac  excitability.     In  our  studies,  three  lines  of  biochemistry  evidence  were  shown  to  confirm   Nav1.5  palmitoylation  in  both  native  expression  background  and  heterologous   expression  system.  Moreover,  palmitoylation  of  Nav1.5  can  be  bidirectionally   regulated  using  2-­Br-­palmitate  and  palmitic  acid.  Our  results  also  demonstrated   that  enhanced  palmitoylation  in  both  cardiomyocytes  and  HEK293  cells   increases  sodium  channel  availability  and  late  sodium  current  activity,  leading  to   enhanced  cardiac  excitability  and  prolonged  action  potential  duration.  In  contrast,   blocking  palmitoylation  by  2-­Br-­palmitiate  increases  closed-­state  channel inactivation  and  reduces  myocyte  excitability.  Our  computer  simulation  results   confirmed  that  the  observed  modification  in  Nav1.5  gating  properties  by  protein   palmitoylation  are  adequate  for  the  alterations  in  cardiac  excitability.  Mutations  of   potential  palmitoylation  sites  predicted  by  CSS-­Palm  bioinformatics  tool  were   introduced  into  wild-­type  Nav1.5  constructs  using  site-­directed  mutagenesis.   Further  studies  revealed  four  cysteines  (C981,  C1176,  C1178,  C1179)  as   possible  Nav1.5  palmitoylation  sites.  In  particular,  a  mutation  of  one  of  these   sites(C981)  is  associated  with  cardiac  arrhythmia  disease.  Cysteine  to   phenylalanine  mutation  at  this  site  largely  enhances  of  channel  closed-­state   inactivation  and  ablates  sensitivity  to  depalmitoylation.  Therefore,  C981  might  be   the  most  important  site  that  regulates  Nav1.5  palmitoylation.  In  summary,  this   dissertation  research  identified  novel  post-­translational  modification  on  Nav1.5   and  revealed  important  details  behind  this  process.  Our  data  provides  new   insights  on  how  post-­translational  lipid  modification  alters  cardiomyocyte   excitability  and  its  potential  role  in  arrhythmogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channels (VGSCs) are critical determinants of neuronal excitability. They set the threshold for action potential generation. The VGSC isoform Nav1.6 participates in various physiological processes and contributes to distinct pathological conditions, but how Nav1.6 function is differentially regulated in different cell types and subcellular locations is not clearly understood. Some VGSC isoforms are subject to S-palmitoylation and exhibit altered functional properties in different S-palmitoylation states. This dissertation investigates the role of S-palmitoylation in Nav1.6 regulation and explores the consequences of S-palmitoylation in modulating neuronal excitability in physiological and pathological conditions. The aims of this dissertation were to 1) provide biochemical and electrophysiological evidence of Nav1.6 regulation by S-palmitoylation and identify specific S-palmitoylation sites in Nav1.6 that are important for excitability modulation, 2) determine the biophysical consequences of epilepsy-associated mutations in Nav1.6 and employ computational models for excitability prediction and 3) test the modulating effects of S-palmitoylation on aberrant Nav1.6 activity incurred by epilepsy mutations. To address these aims, an acyl-biotin exchange assay was used for Spalmitoylation detection and whole-cell electrophysiology was used for channel characterization and excitability examination. The results demonstrate that 1) Nav1.6 is biochemically modified and functionally regulated by S-palmitoylation in an isoform- and site-specific manner and altered S-palmitoylation status of the channel results in substantial changes of neuronal excitability, 2) epilepsy associated Nav1.6 mutations affect different aspects of channel function, but may all converge to gain-of-function alterations with enhanced resurgent currents and increased neuronal excitability and 3) S-palmitoylation can target specific Nav1.6 properties and could possibly be used to rescue abnormal channel function in diseased conditions. Overall, this dissertation reveals S-palmitoylation as a new mechanism for Nav1.6 regulation. This knowledge is critical for understanding the potential role of S-palmitoylation in isoform-specific regulation for VGSCs and providing potential targets for the modulation of excitability disorders. / 2022-05-06

Ras is a central player in signal transduction that mediates cellular proliferation and differentiation. Recent evidence has shown that lipid and non-lipid modified domains participate in Ras traffic and that plasma membrane association is mediated by vectorial vesicular transport from the endomembrane system. ERp57, an ER chaperone, has been shown to specifically bind farnesylated Hras but not non-farnesylated Hras. The objective of this study was to determine if ERp57 participates in Ras trafficking and function. First, the effect of ERp57 knock down by siRNA technology on Hras function was studied; there was a reduction in ERp57 cellular levels that led to a decrease of active ras. Second, specific anti-ERp57 antibodies were delivered into 3T3 cells expressing GFP-ras chimeras to observe the effect on intracellular trafficking. Anti-ERp57 antibodies blocked Hras plasma membrane localization but not Kras suggesting that ERp57 may be involved in Hras intracellular trafficking and function.

Exocytosis, the fusion of a vesicle to a cellular membrane, involves a protein named SNAP-25. This protein, containing two alpha helices connected with a linker region, is localized to the cell membrane via palmitic acids attached to the cysteine residues of its linker region in a process called palmitoylation. Are cysteine residues of the SNAP-25 linker region palmitoylated in an ordered manner and to a particular extent? The answer to this question may give insight into the regulated nature of exocytosis. While it is generally accepted that SNAP-25 must be palmitoylated in order to perform its exocytotic functions, the details surrounding this process are still being discovered, defined, and understood. In these studies we replicate the oxidation, reduction, and palmitoylation of SNAP-25 in vitro. Palmitoylating SNAP-25 in vitro, a process which occurs regularly in vivo, allows us to determine the extent of palmitoylation. In vitro palmitoylation of SNAP-25 was studied both with and without a native palmitoyl acyl transferase (PAT), DHHC-17, the enzyme to attach palmitic acids to cysteines in the linker region of SNAP-25. These studies were done under a variety of conditions designed to identify (1) components necessary for optimal palmitoylation and (2) extent of palmitoylation with components that mimic native conditions. Palmitoylation is a common modification for a variety of proteins, both soluble and membrane-bound. Like phosphorylation, palmitoylation is reversible and may play an important role in regulation of cellular processes. Specifically, the palmitoylation of SNAP-25 may play a critical role in the regulation of exocytosis and therefore learning further details about this important process may help us to better understand a variety of neurodegenerative diseases and states of decreased or compromised exocytosis.

<p>Regulators of G protein signaling (RGS) proteins are key negative regulators of Gα signaling, a branch of G-protein-coupled receptor (GPCR)-mediated signal transduction. Approximately 35% of drugs approved by the Food and Drug Administration (FDA) target GPCRs, so it is not surprising that the discovery of RGS proteins has triggered an interest in them as new drug targets. Even though many studies have been shown the involvement of RGS proteins in cancers, there is still a knowledge gap in understanding function and regulation of RGS proteins in these diseases. Consequently, in this thesis, I explored roles of two RGS proteins that have been implicated in cancers.</p> <p>RGS2 is proposed to act as a tumor suppressor in many different cancers, such as breast cancer, bladder, and ovarian cancer. Here, we investigated if RGS2 also plays a tumor suppressor role in UM, whose growth is driven by overactivated Gαq/11 signaling. We found that increased expression levels of RGS2 inhibit cell growth of UM 92.1 and Mel-202 cells. Mechanistically, this cell growth inhibition is dependent on the association between RGS2 and Gαq, but independent of its canonical GTPase-accelerating protein (GAP) activity. Furthermore, RGS2 inhibited the Mitogen-activated protein kinases (MAPK) signaling, downstream of Gαq, while leaving Yes-associated protein 1/Transcriptional coactivator with PDZ-binding motif (YAP/TAZ) activation unaffected. These data indicate a tumor suppressor role for RGS2 in UM and proposes RGS2 stabilization as a potential therapeutic targeting strategy. </p> <p>In contrast to RGS2, RGS20 contributes to cancer progression, particularly in breast cancer. However, how RGS20 is regulated is understudied. Palmitoylation, a reversible post-translational modification, regulates functions of other RGS proteins, and RGS20 is predicted to be palmitoylated. We provided direct evidence of RGS20 palmitoylation in cells and validated the palmitoylation site as the conserved cysteine (Cys148) in the RGS domain. Our results showed that palmitoylation on this site does not affect its GAP activity and subcellular localization, but it affects the association between RGS20 and active Gαo, and inhibition of Gαo-mediated signaling. This study serves as a foundation for future studies in furthering understating the role of palmitoylation in RGS20 function and its possible implications in cancer biology. </p>

PagP is an integral outer membrane enzyme that transfers a palmitoyl group from a phospholipid to lipid A and the polar headgroup of phosphatidylglycerol (PG). Palmitoyl-lipid A and palmitoyl-PG (PPG) have been implicated in resistance to host immune defenses. PagP proteins are diverse, the E. coli PagP belongs to the major clade of PagP homologs and palmitoylates lipid A regiospecifically at the 2-position, whereas P. aeruginosa PagP belongs to the minor clade of PagP homologs and instead palmitoylates lipid A regiospecifically at the 3’-position. Our objective was to understand how PagP has been adapted in nature to interact with multiple lipid substrates and products. We investigated the structure-function relationships of key major clade homologs, to show that Bordetella PagP palmitoylates lipid A at the 3’-position and employs surface residue T29 in its palmitoyltransferase reaction. Legionella PagP palmitoylates lipid A at the 2-position and was confirmed to select a palmitate chain from a pool including iso-methyl branched phospholipids characteristic of this species. PagP is usually encoded as a single copy on the chromosome in most bacteria, but two copies of pagP are found in endophytic bacteria. These duplicated PagP homologs from the major clade branch into two subclades, namely chromosomal and plasmid-based PagP homologs. The chromosomal PagP homologs exhibit interacting periplasmic D61 and H67 residues, which are naturally mutated in plasmid-based PagP homologs, and are associated with a conformational change in the -barrel that determines its ability to palmitoylate PG. Chromosomal PagPs can convert PPG to bis(monoacylglycero)phosphate (BMP) and lysophosphatidylglycerol (LPG) through a periplasmic active site controlled by the invariant Y87 residue of E. coli PagP. Plasmid-based PagP homologs appear to have been adapted instead as monofunctional lipid A palmitoyltransferases. These results points to a common ancestor for PagP proteins. Knowledge gained from these studies can be applied to protein engineering. / Thesis / Doctor of Philosophy (PhD)

<p>The spectrin-ankyrin network assembles diverse plasma membrane domains including axon initial segments and nodes of Ranvier, cardiomyocyte T-tubules and intercalated discs, epithelial lateral membranes, costameres and photoreceptor inner and outer segments. However the mechanism that targets the spectrin-ankyrin network to those plasma membrane domains is unknown. This thesis identifies two lipid inputs from protein palmitoylation and phosphoinositides that together control the precise localization of the spectrin-ankyrin network. In Chapter 2, we identify a linker peptide encoded by a single divergent exon that distinguishes the subcellular localization of ankyrin-B and -G by selectively suppressing protein binding through autoinhibition. In Chapter 3, we demonstrate that ankyrin-G is S-palmitoylated at a conserved C70 residue which is required to assemble epithelial lateral membranes and neuronal axon initial segments. We continue to interrogate how palmitoylation regulates ankyrin-G activities in Chapter 4, and identify DHHC5 and DHHC8 as the palmitoyltransferases in MDCK cells. We showed that palmitoylated ankyrin-G, in concert with phosphoinositide lipids, determines the polarized localization of beta II spectrin though a coincidence detection mechanism. This palmitoyltransferases/ ankyrin-G/beta II spectrin pathway determines the cell height of columnar epithelial cells. In Chapter 5, we elucidated the molecular mechanism through which the spectrin-ankyrin network assembles epithelial lateral membranes. We demonstrated that ankyrin-G and beta II spectrin function by opposing clathrin-mediated endocytosis to build the lateral membrane in MDCK cells. Together, this thesis dissects the mechanisms of how the spectrin-ankyrin network achieves precise membrane targeting and how it assembles lateral membranes to determine the morphogenesis of columnar epithelial cells, and provides the first molecular insight to understand how cells control the assembly of diverse plasma membrane domains.</p> / Dissertation

Protein palmitoylation refers to the process of adding a 16-carbon saturated fatty acid to the cysteine of a substrate protein, and this can in turn affect the substrate’s localization, stability, folding, and several other processes. This process is catalyzed by a family of 23 mammalian protein acyltransferases (PATs), a family of transmembrane enzymes that modify an estimated 10% of the proteome. At this point in time, no structure of a protein in this family has been solved, and therefore there is poor understanding about the regulation of the enzymes and their substrates. Most proteins, including palmitoylation enzymes and substrates, have some level of intrinsic disorder, and this flexibility can be important for signaling processes such as protein- protein interactions and post-translational modifications. Therefore, we assumed that examining intrinsic disorder in palmitoylation enzymes and substrates would yield insight into their regulatory mechanisms. First, we found that among other factors, utilizing intrinsic disorder predictions led to a palmitoylation predictor that significantly outperformed existing predictors. Next, we discovered a conserved region of predicted disorder-to-order transition in the disordered C-termini of the PAT family. In Erf2, the yeast Ras PAT, we developed a model where this region reversibly interacts with membranes, and we found that this region mediates interaction with Acc1, an enzyme involved in fatty acid metabolism processes. Finally, we found that an XLID-associated nonsense mutation in zDHHC9, the mammalian Ras PAT, removed a disordered region that was critical for enzyme localization. Future studies of palmitoylation utilizing the framework of intrinsic disorder may lead to additional insights about this important regulatory process.

Neurons depend upon neurotransmitter release through regulated exocytosis to accomplish the immense processing performed within the central nervous system. The SNARE hypothesis points to a family of proteins that are thought to enable the membrane fusion that leads to exocytosis. The secondary structure of SNAP-25 is unique among SNARE proteins in that it has two alpha helical SNARE motifs and a cysteine rich (C85, C88, C90, C92) membrane interacting region but notransmembrane domain. The cysteines may be modified by palmitoylation or oxidation but the role of these modifications in vivo is not well understood. Our goal is to elucidate possible regulatory roles of SNAP-25 that relate to its unique structure and these reversible modifications. However, the study of SNAP-25 in reconstituted systems is hampered by a lack of readily available palmitoylated SNAP-25. A method for in vitro palmitoylation of SNAP-25 by HIP14, a neuronal acyltransferase, is described along with the application of a biotinylation streptavidin assay to verify palmitoylation. Palmitoylation increases the extent to which SNAP-25 interacts with lipids as observed with an environment sensitive trpytophan fluorescence assay. Palmitoylation also alters the phase transition of DPPC lipids differently than unpalmitoylated SNAP-25.This effect on the membrane may influence fusion events. Oxidation of the cysteine residues may be responsible for the sensitivity of SNAP-25 to reactive oxygen species. Our data suggests that, when oxidized, SNAP-25 does not interact with membranes to the same extent as palmitoylated SNAP-25. This may provide a mechanism for reducing exocytosis during oxidative stress. Also, oxidized SNAP-25 is not susceptible to Botulinum Neurotoxin E. The effects of oxidation and palmitoylation on the protein interactions of SNAP-25 may shed light on its role in the SNARE complex and membrane fusion.