Browsing by Author "McNew, James A."
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Item A structure-function characterization of the ER membrane protein atlastin(2012) Moss, Tyler J.; McNew, James A.The biogenesis and maintenance of the entire endomembrane system is dependent upon membrane fusion proteins. Mounting evidence indicates that the integral membrane GTPase Atlastin is a membrane fusion protein involved in the homotypic fusion of the endoplasmic reticulum (ER) membrane suggesting a role in the biogenesis and maintenance of ER structure. I helped show that recombinant Drosophila atlastin is able to promote the fusion of synthetic membranes in vitro and that this fusion is dependent upon atlastin GTPase activity. The structure-function experiments presented here assist in elucidating domains required in the mechanism of atlastin mediated membrane fusion. ER homotypic fusion is dependent upon the self-association of Atlastin subunits in adjacent membranes to bring the bilayers into close molecular contact. Atlastin dimerization occurs in the presence of GTPγS but not GDP and stable dimerization is dependent upon a juxtamembrane middle domain three-helix bundle (3HB). The atlastin GTPase domain and 3HB form a potent soluble domain inhibitor of atlastin homotypic fusion, while the GTPase domain alone shows little inhibition. Designed GTPase domain mutations show that GTP binding and atlastin dimerization is insufficient to support fusion without GTP hydrolysis. Additionally, domain analysis of atlastin reveals that the C-terminal cytoplasmic domain of atlastin is absolutely required for membrane fusion, possibly through a protein-lipid interaction of an amphipathic alpha-helix. Genetic lesions in the human Atlastin-1 gene, SPG3A, result in a form of autosomal dominant hereditary spastic paraplegia (HSP). A better understanding of Atlastin function should lend significant insight into normal ER biogenesis and maintenance, as well as the pathology of human disease.Item Attack on single Escherichia coli spheroplast by antimicrobial peptides(2015-08-13) Sun, Tzu-Lin; Huang, Huey W.; Hafner, Jason H.; McNew, James A.Studies of the molecular mechanisms of antimicrobial peptides (AMPs) have mostly been performed with lipid bilayers, as a substitute for cell membrane. Hence, there is a persistent question as to whether the action of AMPs on bacterial membranes can be reproduced on lipid bilayers. Valuable information was obtained recently from observing the actions of AMPs on E. coli and Bacillus subtilis by time-lapse fluorescence microscopy. The goal of my dissertation is to study the direct action of AMPs on the cytoplasmic membranes by using E. coli spheroplasts, the cell form from which the outer membranes have been removed. The key question is how to reveal the response of the spheroplast to AMPs. In our previous work, the aspiration method on giant unilamellar vesicle (GUV) has been demonstrated as very effective for researching membrane effects induced by peptides. This method is able to measure the membrane expansion due to peptide binding and simultaneously monitor membrane permeability by using dye indicators. In this work, a spray method was developed for introducing AMPs and customized the experimental procedures for performing the experiments of E. coli spheroplasts. The living state of cytoplasmic membrane makes it difficult to deduce the molecular events from the response of live cells. Hence, the physical methods which have been developed for researching peptide activity on lipid bilayers were practiced first. These methods include X-ray diffraction (XRD), oriented circular dichroism (OCD) and GUV aspiration. These methods were practiced by studying the question as to why a hydrocarbon-stapled peptide NYAD-1 drug was reported to have membrane permeating property. Melittin was selected as the representative of AMPs for the E. coli spheroplast experiments. To better understand the characteristics of melittin, the melittin activity on model lipid membrane was examined before advancing to the spehroplast experiment. The melittin transmembrane was proposed by correlating melittin binding on a lipid vesicle (aspirated GUV imaging) with structural studies in multilayers (XRD and OCD). This behavior was further determined by using fluorescence indicator to track the melittin distribution. The toroidal structure of melittin pore was also detected by grazing-angle X-ray anomalous diffraction. In the studies of NAYD-1 and melittin on a model membrane, our discovery of AMP's transmembrane enables us to clarify the pore-formation mechanism which has been disputed for decades. In addition, our past work on the physical property of E. coli spheroplast cytoplasmic membrane has indicated the existence of a lipid reservoir. The lipid reservoir dominates the surface tension by balancing the membrane folds. Finally, we used the aspiration method to hold a spheroplast so as to measure the change of the spheroplast membrane area in response to the AMPs' binding. Further, a fluorescence indicator which is able to associate with AMPs was used to monitor the peptide distribution and another fluorescence dye to monitor the molecule leakage. The spheroplast study shows that there are similarities and differences between the responses of spheroplasts and GUVs. The recent findings on the unique properties of spheroplast membranes are the key for understanding these results. Our work of understanding the AMPs activity on membrane is potentially applicable in improving peptide drug design and delivery for disease treatment.Item Characterization of Proteins Involved in Membrane Fusion- Atlastin and Munc18c(2013-09-16) Verma, Avani; McNew, James A.; Braam, Janet; Raphael, Robert M.; Shamoo, Yousif; Wagner, Daniel S.Membranes provide a barrier to cells and organelles, and allow the selective transport of molecules between compartments. Membrane fusion is essential for organelle biogenesis as well as trafficking of molecules between cellular compartments. Membrane fusion is also required for the formation of the branched network of tubules that make up the Endoplasmic Reticulum (ER). One protein implicated in ER fusion is Atlastin, a dynamin like GTPase. Mutations in Atlastin-1, among others, cause Hereditary Spastic Paraplegias (HSP), a group of neurological disorders that cause progressive weakness of lower extremities. We have shown that the C-terminal tail of atlastin is necessary for membrane fusion. The requirement of the C-terminal tail can be partially abrogated in an unstable lipid environment. This implies that the C-terminal tail of Atlastin plays a role in perturbing the lipid bilayer to allow membrane fusion. Understanding the molecular details of how Atlastin drives membrane fusion may help elucidate the pathogenesis of HSP. Intracellular fusion at the plasma membrane is SNARE mediated and regulated by Sec1p/Munc18 (SM) proteins. Increased rate of glucose transport into fat and muscles cells by translocation of glucose transporter GLUT4 in response to insulin is a SNARE regulated fusion process. Recent reports have linked Munc18c and Syntaxin4 with obesity and Type 2 diabetes. We characterized the function of Munc18c, an SM protein, in regulating GLUT-4 containing vesicle fusion with the plasma membrane. We have shown that Munc18c directly inhibits membrane fusion by interacting with its cognate SNARE complexes. Characterization of membrane fusion in a minimal system as the in vitro liposome fusion assay offers a powerful tool with which to finely dissect the mechanistic basis of SM protein function.Item Characterization of Structure and Function Relationship between Domains of the ER Membrane Protein Atlastin(2014-02-06) Desai, Tanvi; McNew, James A.; Huang, Huey W.; Braam, Janet; Stern, Michael; Lwigale, Peter YunjuThe endoplasmic Reticulum (ER) is an important site for lipid synthesis, protein synthesis and transport. ER fusion is an essential process for its maintenance and biogenesis. Mutations in genes involved in this process cause Hereditary Spastic Paraplegia (HSP). These mutations are shown to affect intracellular trafficking and localization of membrane compartment. One of the important proteins causing early onset of HSP is Atlastin. Previous work in McNew lab at Rice University (Moss et al., 2011b) has shown that atlastin is involved in the homotypic fusion of the ER and the C-terminal cytoplasmic region of atlastin is essential for atlastin mediated fusion. During my studies presented in this thesis, I was able to demonstrate that the C-terminal cytoplasmic region of atlastin destabilizes lipid bilayers to facilitate fusion. The requirement of C-terminal cytoplasmic region is minimal when fusing two fluid (or unstable) lipid bilayers. The C-terminal cytoplasmic region of atlastin forms an amphipathic helix and mutations on the hydrophobic phase of the helix reduce fusion. These mutations are not dominant, as presence of full length atlastin on even one of the fusing lipid bilayers can significantly improve fusion during a heterotypic fusion reaction. Additionally, domain swaps between human atlastin-1 and drosophila atlastin show that the role of C-terminal cytoplasmic region is highly conserved. Also, during my research presented here in, I found that when the transmembrane region and C-terminal cytoplasmic region of human atlastin-1 were swapped with drosophila atlastin, it showed functional similarity. These results show that although atlastins in organisms play an important role in the ER fusion, there are likely species specific differences in how this is achieved. An understanding of atlastin mediated fusion should help in unraveling mechanisms of HSP pathogenesis and other disorders arising from dysfunctional ER.Item Characterization of the stimulation of SNARE-mediated membrane fusion by the SM protein Munc18a(2009) Rodkey, Travis Lincoln; McNew, James A.The mammalian neuronal Sec1p/Munc18 protein Muncl8a binds tightly to the t-SNARE Syntaxin1a in isolation, and the binary Muncl8a/Syntaxin1a interaction is thought to prevent formation of the Syntaxin1a/SNAP25b t-SNARE complex required for intracellular membrane fusion. However, both Sec1p and Munc18a are required factors for exocytosis and stimulate in vitro membrane fusion of the appropriate preassembled t-SNARE complex and v-SNARE. The experiments presented here show that Muncl8a stimulates the initial rate and final extent of membrane fusion driven by the neuronal t-SNAREs Syntaxin1a/SNAP25b and v-SNARE VAMP2 and elucidate the molecular mechanism underlying the increase in membrane fusion. Munc18a binds to the assembled neuronal t-SNARE complex in a functional manner much like its yeast homolog Sec1p. The Munc18a/t-SNARE complex interaction appears to occur through Syntaxin1a but not SNAP25b because Munc18a stimulates fusion of a Syntaxin1a/yeast Sec9c t-SNARE complex and fails to interact with yeast SNAREs. Munc18a displays a well-characterized interaction with the Syntaxin1a N-terminal regulatory domain (NRD), but also contacts the Syntaxin1a SNARE Core Complex (H3) domain to stimulate fusion because fusion of a yeast t-SNARE complex chimera in which the yeast Sso1p NRD was replaced with the Syntaxin1a NRD was not stimulated by Munc18a. Deletion of a flexible linker region between the Syntaxin1a NRD and H3 domain that permits movement of the NRD abolishes the ability of Munc18a to stimulate membrane fusion, suggesting that Munc1a may actively position the Syntaxin1a NRD to favor membrane fusion. In addition, Munc18a directly and functionally interacts with VAMP2. Taken together, these results suggest strongly that Munc18a acts as a scaffold that increases the efficiency of t-SNARE complex and v-SNARE interactions, resulting in accelerated membrane fusion. Furthermore, Munc18a may also function in combination with the calcium sensor Synaptotagmin I to enhance neurosecretion at the last stage of exocytosis.Item Enhanced sampling and applications in protein folding(2013-07-24) Zhang, Cheng; Ma, Jianpeng; McNew, James A.; Igoshin, Oleg A.We show that a single-copy tempering method is useful in protein-folding simulations of large scale and high accuracy (explicit solvent, atomic representation, and physics-based potential). The method uses a runtime estimate of the average potential energy from an integral identity to guide a random walk in the continuous temperature space. It was used for folding three mini-proteins, trpzip2 (PDB ID: 1LE1), trp-cage (1L2Y), and villin headpiece (1VII) within atomic accuracy. Further, using a modification of the method with a dihedral bias potential added on the roof temperature, we were able to fold four larger helical proteins: α3D (2A3D), α3W (1LQ7), Fap1-NRα (2KUB) and S-836 (2JUA). We also discuss how to optimally use simulation data through an integral identity. With the help of a general mean force formula, the identity makes better use of data collected in a molecular dynamics simulation and is more accurate and precise than the common histogram approach.Item Human astrovirus capsid protein releases a membrane lytic peptide upon trypsin maturation(American Society for Microbiology, 2023) Ykema, Matthew; Ye, Kai; Xun, Meng; Harper, Justin; Betancourt-Solis, Miguel A.; Arias, Carlos F.; McNew, James A.; Tao, Yizhi JaneThe human astrovirus (HAstV) is a non-enveloped, single-stranded RNA virus that is a common cause of gastroenteritis. Most non-enveloped viruses use membrane disruption to deliver the viral genome into a host cell after virus uptake. The virus–host factors that allow for HAstV cell entry are currently unknown but thought to be associated with the host-protease-mediated viral maturation. Using in vitro liposome disruption analysis, we identified a trypsin-dependent lipid disruption activity in the capsid protein of HAstV serotype 8. This function was further localized to the P1 domain of the viral capsid core, which was both necessary and sufficient for membrane disruption. Site-directed mutagenesis identified a cluster of four trypsin cleavage sites necessary to retain the lipid disruption activity, which is likely attributed to a short stretch of sequence ending at arginine 313 based on mass spectrometry of liposome-associated peptides. The membrane disruption activity was conserved across several other HAstVs, including the emerging VA2 strain, and effective against a wide range of lipid identities. This work provides key functional insight into the protease maturation process essential to HAstV infectivity and presents a method to investigate membrane penetration by non-enveloped viruses in vitro.Item Intra- and inter-cellular membrane fusion mechanisms in Saccharomyces cerevisiae(2008) Liu, Song; McNew, James A.Intra- and inter-cellular membrane fusion processes are all mediated by specialized membrane fusion proteins and baker's yeast Saccharomyces cerevisiae has been an invaluable model system to study these fusion events. Intracellular fusion in yeast is mediated by a large family of proteins named SNAREs, however, the molecular machinery mediating intercellular membrane fusion between yeast haploid cells remains unknown. Despite the identification of SNAREs as the minimal membrane fusion machinery for yeast intracellular fusion, additional regulatory mechanisms remain to be elucidated. Sec9p and Spo20p are two SNAP25 family SNAREs specialized for different developmental stages in yeast. Sec9p interacts with Sso1/2p and Snc1/2p to mediate membrane fusion between post-Golgi vesicles and the plasma membrane during vegetative growth. Spo20p replaces Sec9p in the generation of prospore membranes during sporulation. The function of Spo20p requires phosphatidic acid (PA) to be generated in the prospore membrane, the reason of which has not been fully understood. I compared the in vitro membrane fusion function of Sec9p and Spo20p and found that Spo20p forms a less fusion efficient SNARE complex than Sec9p. Incorporation of PA in the lipid bilayer stimulates Sec9p or Spo20p mediated membrane fusion, likely by decreasing the energetic barrier during membrane merger. I also found that PA could directly interact with the positively charged juxtamembrane region of Sso1p, which contributed to the stimulatory effects of PA as well. These results suggested that the fusion strength of SNAREs, the composition of organelle lipids and lipid-SNARE interactions could be coordinately regulated to control the rate and specificity of membrane fusion. I also tried to identify the fusogens mediating yeast intercellular fusion using proteomic methods. We hypothesized that such fusogens were induced by mating pheromone and transported to the cell-cell fusion sites. I was able to isolate post-Golgi vesicles from yeast cells stimulated with mating pheromone and find yeast intercellular fusion related proteins associated with these vesicles. However, our proteomic analysis of the proteins on these vesicles failed to identify any fusogen candidates.Item Munc18b is an essential gene in mice whose expression is limiting for secretion by airway epithelial and mast cells(Portland Press Limited, 2012) Kim, Kyubo; Petrova, Youlia M.; Scott, Brenton L.; Nigam, Rupesh; Agrawal, Anurag; Evans, Christopher M.; Azzegagh, Zoulikha; Gomez, Alejandra; Rodarte, Elsa M.; Olkkonen, Vesa M.; Bagirzadeh, Rustam; Piccotti, Lucia; Ren, Binhui; Yoon, Joo-Heon; McNew, James A.; Adachi, Roberto; Tuvim, Michael J.; Dickey, Burton F.; Cystic Fibrosis Foundation; National Institutes of HealthAirway mucin secretion and MC (mast cell) degranulation must be tightly controlled for homoeostasis of the lungs and immune system respectively. We found the exocytic protein Munc18b to be highly expressed in mouse airway epithelial cells and MCs, and localized to the apical pole of airway secretory cells. To address its functions, we created a mouse with a severely hypomorphic Munc18b allele such that protein expression in heterozygotes was reduced by∼50%. Homozygous mutant mice were not viable, but heterozygotes showed a ∼50% reduction in stimulated release of mucin from epithelial cells and granule contents from MCs. The defect in MCs affected only regulated secretion and not constitutive or transporter-mediated secretion. The severity of passive cutaneous anaphylaxiswas also reduced by ∼50%, showing that reduction of Munc18b expression results in an attenuation of physiological responses dependent on MC degranulation. The Munc18b promoter is controlled by INR (initiator), Sp1 (specificity protein 1), Ets, CRE (cAMP-response element), GRE (glucocorticoid-response element), GATA and E-box elements in airway epithelial cells; however, protein levels did not change during mucous metaplasia induced by allergic inflammation. Taken together, the results of the present study identifyMunc18b as an essential gene that is a limiting component of the exocytic machinery of epithelial cells and MCs.Item Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse(Public Library of Science, 2017) Wangler, Michael F.; Chao, Yu-Hsin; Bayat, Vafa; Giagtzoglou, Nikolaos; Shinde, Abhijit Babaji; Putluri, Nagireddy; Coarfa, Cristian; Donti, Taraka; Graham, Brett H.; Faust, Joseph E.; McNew, James A.; Moser, Ann; Sardiello, Marco; Baes, Myriam; Bellen, Hugo J.Peroxisome biogenesis disorders (PBD) are a group of multi-system human diseases due to mutations in the PEX genes that are responsible for peroxisome assembly and function. These disorders lead to global defects in peroxisomal function and result in severe brain, liver, bone and kidney disease. In order to study their pathogenesis we undertook a systematic genetic and biochemical study of Drosophila pex16 and pex2 mutants. These mutants are short-lived with defects in locomotion and activity. Moreover these mutants exhibit severe morphologic and functional peroxisomal defects. Using metabolomics we uncovered defects in multiple biochemical pathways including defects outside the canonical specialized lipid pathways performed by peroxisomal enzymes. These included unanticipated changes in metabolites in glycolysis, glycogen metabolism, and the pentose phosphate pathway, carbohydrate metabolic pathways that do not utilize known peroxisomal enzymes. In addition, mutant flies are starvation sensitive and are very sensitive to glucose deprivation exhibiting dramatic shortening of lifespan and hyperactivity on low-sugar food. We use bioinformatic transcriptional profiling to examine gene co-regulation between peroxisomal genes and other metabolic pathways and we observe that the expression of peroxisomal and carbohydrate pathway genes in flies and mouse are tightly correlated. Indeed key steps in carbohydrate metabolism were found to be strongly co-regulated with peroxisomal genes in flies and mice. Moreover mice lacking peroxisomes exhibit defective carbohydrate metabolism at the same key steps in carbohydrate breakdown. Our data indicate an unexpected link between these two metabolic processes and suggest metabolism of carbohydrates could be a new therapeutic target for patients with PBD.Item Peroxisome Biogenesis in Drosophila melanogaster: Protein Trafficking, Lipid Metabolism, and Muscle Function(2013-12-02) Faust, Joseph; McNew, James A.; Bartel, Bonnie; Diehl, Michael R.; Stern, Michael; Bennett, Matthew R.Peroxisomes are ubiquitous organelles required for many essential functions, such as fatty acid metabolism. Defects in peroxisome biogenesis cause a spectrum of human diseases known as peroxisome biogenesis disorders (PBDs). These devastating diseases lack effective therapies and it is unclear how peroxisome dysfunction causes the disease state. Animal models are needed to understand the connection between peroxisome biology and animal physiology. The fruit fly, Drosophila melanogaster, has recently become an important animal model in the study of peroxisomes. We have identified the major peroxisomal proteins and pathways in flies and examined peroxisomal protein trafficking. We have found that fruit fly peroxisomes share many features in common with higher animals, but display some important differences. Flies appear to have lost one of the pathways used in other organisms to target proteins to the peroxisomal matrix. Also some proteins are dually localized to peroxisomes and the cytoplasm likely through a weak interaction with the protein machinery that brings peroxisomal proteins into the organelle. We have also generated fly mutants with impaired peroxisome biogenesis and shown that peroxisomes are required for normal development and lipid metabolism. Flies with impaired peroxisome biogenesis also show defects in multiple processes that depend on muscle function, such as locomotion. PBD patients also display muscle defects, but it is thought to be a secondary effect of neuronal dysfunction. We propose that peroxisome loss in humans, like in flies, may directly affect muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in fly physiology and specifically in muscle cells may reveal novel aspects of PBD etiology.Item Peroxisomes Are Required for Lipid Metabolism and Muscle Function in Drosophila melanogaster(Public Library of Science, 2014) Faust, Joseph E.; Manisundaram, Arvind; Ivanova, Pavlina T.; Milne, Stephen B.; Summerville, James B.; Brown, H.Alex; Wangler, Michael F.; Stern, Michael; McNew, James A.Peroxisomes are ubiquitous organelles that perform lipid and reactive oxygen species metabolism. Defects in peroxisome biogenesis cause peroxisome biogenesis disorders (PBDs). The most severe PBD, Zellweger syndrome, is characterized in part by neuronal dysfunction, craniofacial malformations, and low muscle tone (hypotonia). These devastating diseases lack effective therapies and the development of animal models may reveal new drug targets. We have generated Drosophila mutants with impaired peroxisome biogenesis by disrupting the early peroxin gene pex3, which participates in budding of pre-peroxisomes from the ER and peroxisomal membrane protein localization. pex3 deletion mutants lack detectible peroxisomes and die before or during pupariation. At earlier stages of development, larvae lacking Pex3 display reduced size and impaired lipid metabolism. Selective loss of peroxisomes in muscles impairs muscle function and results in flightless animals. Although, hypotonia in PBD patients is thought to be a secondary effect of neuronal dysfunction, our results suggest that peroxisome loss directly affects muscle physiology, possibly by disrupting energy metabolism. Understanding the role of peroxisomes in Drosophila physiology, specifically in muscle cells may reveal novel aspects of PBD etiology.Item Regulation of neuronal SNARE complexes by complexin and calcium(+2)-synaptotagmin(2008) Doneske, Blair; McNew, James A.Synaptic transmission relies on an exquisitely orchestrated series of protein-protein interactions that ultimately results in neurotransmitter release into the synaptic cleft. The final step in this reaction is membrane fusion of the synaptic vesicle with the presynaptic plasma membrane catalyzed by SNARE proteins. Several extrinsic protein factors impinge on the cycle of SNARE complex assembly to regulate vesicle release in space and time. These include general regulators such as the Rab family of GTPases and the Munc18/Sect (SM) proteins, as well as specific regulators of synaptic transmission including complexin and synaptotagmin. The interplay of these regulatory factors and their precise mechanisms of action remain an area of intense investigation. This work shows that fusion driven by a t-SNARE complex of Syntaxin1A/SNAP25 and the v-SNARE VAMP2, but not their yeast equivalents, is inhibited by complexin. Furthermore, inner leaflet mixing is strongly impaired relative to total lipid mixing indicating that inhibition by complexin arrests fusion at a hemifusion intermediate. When the calcium sensor synaptotagmin is added in the absence of calcium to the complexin-inhibited reaction, the arrest persists. However, when calcium is introduced, complexin inhibition is relieved and full fusion rapidly proceeds as evidenced by restoration of inner leaflet mixing. These results suggest that the combination of complexin and synaptotagmin provides a strong calcium-dependent clamp to inhibit full fusion at hemifusion until released by calcium influx.Item Regulation of SNARE-mediated membrane fusion by Sec1/Munc18 (SM) proteins(2005) Scott, Brenton L.; McNew, James A.The intricate temporal and spatial regulation of membrane fusion is critical for all living organisms. Fusion of two opposing membranes occurs in a wide range of processes. These include intracellular transportation, cell-to-cell fusion and viral fusion. In all known cases, the SNARE proteins (Soluble NSF attachment protein receptors) (Sollner et al., 1993; Whiteheart et al., 1993) have been shown to be required for vesicular membrane fusion within cells and sufficient to drive membrane fusion in vitro (Nickel et al., 1999; Parlati et al., 1999; Weber et al., 1998). While SNAREs combine in specific combinations to drive highly specific membrane fusion, it is clear that SNARE proteins do not act independently to regulate the entire fusion process. Many regulatory proteins from different families have been identified that interact with individual SNARE proteins and SNARE complexes, yet the precise role of many of these remains unclear. One such group of regulatory proteins is the Sec1/Munc18 (SM) family. Sec1 proteins are likely to be critical players in membrane trafficking. My work has focused on the role of the yeast Sec1p in post-Golgi secretion in Saccharomyces cerevisiae. To analyze Sec1p function in vitro, I have utilized a well-characterized SNARE-mediated membrane fusion assay. For this application, conditions were optimized to allow for specific protein-protein interactions to be tested. Conditions for expression and purification of the previously elusive recombinant Sec1p are documented. In addition, an overexpressing Sec1p yeast strain was generated. Sec1p interactions with SNARE proteins that mediate post-Golgi secretion were then tested. I found that recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p;Sec9c) as well as to the fully assembled ternary-SNARE complex (Sso1p;Sec9c/Snc2p), and also weakly to free Sso1p. I tested the ability of Sec1p to regulate fusion in the fusion assay. Concentration dependent stimulation of membrane fusion is observed when Sec1p is associated with the SNARE proteins. The binding and fusion data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion. With this new information, specific binding modes of neuronal-Sec1 are currently being investigated further in yeast, Drosophila and mammalian SNARE systems.Item Regulation of Syntaxin3B-Mediated Membrane Fusion by T14, Munc18, and Complexin(MDPI, 2023) Nishad, Rajkishor; Betancourt-Solis, Miguel; Dey, Himani; Heidelberger, Ruth; McNew, James A.Retinal neurons that form ribbon-style synapses operate over a wide dynamic range, continuously relaying visual information to their downstream targets. The remarkable signaling abilities of these neurons are supported by specialized presynaptic machinery, one component of which is syntaxin3B. Syntaxin3B is an essential t-SNARE protein of photoreceptors and bipolar cells that is required for neurotransmitter release. It has a light-regulated phosphorylation site in its N-terminal domain at T14 that has been proposed to modulate membrane fusion. However, a direct test of the latter has been lacking. Using a well-controlled in vitro fusion assay, we found that a phosphomimetic T14 syntaxin3B mutation leads to a small but significant enhancement of SNARE-mediated membrane fusion following the formation of the t-SNARE complex. While the addition of Munc18a had only a minimal effect on membrane fusion mediated by SNARE complexes containing wild-type syntaxin3B, a more significant enhancement was observed in the presence of Munc18a when the SNARE complexes contained a syntaxin3B T14 phosphomimetic mutant. Finally, we showed that the retinal-specific complexins (Cpx III and Cpx IV) inhibited membrane fusion mediated by syntaxin3B-containing SNARE complexes in a dose-dependent manner. Collectively, our results establish that membrane fusion mediated by syntaxin3B-containing SNARE complexes is regulated by the T14 residue of syntaxin3B, Munc18a, and Cpxs III and IV.Item Regulatory regions of Sso1p and their roles in SNARE mediated membrane fusion(2005) Van Komen, Jeffrey S.; McNew, James A.Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p) and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion in the secretory pathway, many aspects of SNARE assembly and regulation are ill defined. I examined the yeast regulatory protein, Sec1p, and its function in exocytosis. I show that the majority of Sec1p localizes to the plasma membrane, even though it is predicted to be a soluble protein. Furthermore, a significant portion of Sec1p colocalizes with Sso1, but is enriched in the bud neck. Sec1p binds to the t-SNARE complex and directly stimulates membrane fusion in vitro. I have also examined several defined structural regions of the yeast plasma membrane t-SNARE component Sso1p for their effect on membrane fusion in vivo and in vitro. To analyze the role of the N-terminal regulatory domain in Sso1p, I generated a chimeric protein that physically links the two separate proteins of the yeast plasma membrane t-SNARE complex, namely a truncated Sec9p and Sso1p. With this chimera, I have shown that the required function of the N-terminal regulatory domain Sso1p can be circumvented when t-SNARE complex formation is made intramolecular, suggesting the N-terminal regulatory domain is required for efficient t-SNARE complex formation and does not recruit necessary scaffolding factors. Next, I used targeted sequence modification, including insertions and replacements, in a conserved, highly charged juxtamembrane region between the transmembrane helix and the core coiled-coil domain of Sso1p. The effects of the modifications were examined both in vitro and in vivo. I found that mutant Sso1 proteins with insertions or duplications show limited function in vivo, whereas replacement of as few as three amino acids preceding the transmembrane domain results in a non-functional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting helical continuity between the transmembrane domain and the core coiled-coil domain is not essential. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrate that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.Item Structural and functional studies of the envelope protein and viral polymerase in Hepatitis B Virus(2021-10-18) Lin, Zihan; McNew, James A.; Shamoo, Yousif; Lwigale, Peter; Suh, JunghaeHepatitis B Virus (HBV), a member of the Hepadnaviridae family, is a serious pathogen that causes chronic Hepatitis B and affects over 350 million people globally. Though a vaccine has been available since 1982, it cannot cure established infections and their effectiveness in preventing blood-borne transmission from an infected mother to her infant is only 90%. In order to better understand human HBV with the aim of finding effective treatments, much work has been dedicated toward investigating HBV virally encoded proteins. Unfortunately, few of these proteins have been able to be expressed or purified in vitro. As a result, current efforts seek to determine candidate HBV surface antigens as a possible immunotherapeutic target. Particularly promising candidates include those with identical function as HBV envelope L protein, which interacts with host proteins such as sodium-taurocholate cotransporters polypeptide (NTCP) and possibly epidermal growth factor receptor (EGFR) for entry. Meanwhile, current FDA-approved drugs to control human HBV target the viral polymerase. In order to develop more effective drugs against HBV infection, a better understanding of the HBV polymerase structure and function is necessary. Such information would not only inform us about the enzymatic mechanism of the viral polymerase, but also provide insights into the mechanism of current and mutation-mediated drug resistance. Here, we report the expression, purification, and analysis of the HBV LM envelope protein – a promising surface antigen. Lone LM was found to be an intrinsically disordered monomeric protein capable of interaction with host protein NTCP - and possibly EGFR. Addition of single-chain variable fragment 2H5 to LM formed a much more stable complex while conserving biological activity. Our results overall suggest that LM and the 2H5-LM complex exhibit favorable physical and biochemical properties for future in vitro study, in addition to serving a key role in promoting HBV entry and infection. We also demonstrate the expression and purification of the RNA dependent RNA polymerase domain and RNase H viral polymerase domains from different HBV species.Item The Atlastin C-terminal Tail is an Amphipathic Helix that Perturbs Bilayer Structure during Endoplasmic Reticulum Homotypic Fusion(American Society for Biochemistry and Molecular Biology, 2015) Faust, Joseph E.; Desai, Tanvi; Verma, Avani; Ulengin, Idil; Sun, Tzu-Lin; Moss, Tyler J.; Betancourt, Miguel A.; Huang, Huey W.; Lee, Tina; McNew, James A.Fusion of tubular membranes is required to form three-way junctions found in reticular subdomains of the endoplasmic reticulum (ER). The large GTPase Atlastin has recently been shown to drive ER membrane fusion and three-way junction formation. The mechanism of Atlastin-mediated membrane fusion is distinct from SNARE-mediated and many details remain unclear. In particular, the role of the amphipathic C-terminal tail of Atlastin is still unknown. We have found that a peptide corresponding to the Atlastin C-terminal tail binds to membranes as a parallel alpha helix, induces bilayer thinning, and increases acyl chain disorder. The function of the C-terminal tail is conserved in human Atlastin. Mutations in the C-terminal tail decrease fusion activity in vitro, but not GTPase activity, and impair Atlastin function in vivo. In the context of unstable lipid bilayers, the requirement for the C-terminal tail is abrogated. These data suggest that the C-terminal tail of Atlastin locally destabilizes bilayers to facilitate membrane fusion.Item The Drosophila melanogaster attP40 docking site and derivatives are insertion mutations of msp-300(Public Library of Science, 2022) Graaf, Kevin van der; Srivastav, Saurabh; Singh, Pratibha; McNew, James A.; Stern, MichaelThe ɸC31 integrase system is widely used in Drosophila melanogaster to allow transgene targeting to specific loci. Over the years, flies bearing any of more than 100 attP docking sites have been constructed. One popular docking site, termed attP40, is located close to the Nesprin-1 orthologue msp-300 and lies upstream of certain msp-300 isoforms and within the first intron of others. Here we show that attP40 causes larval muscle nuclear clustering, which is a phenotype also conferred by msp-300 mutations. We also show that flies bearing insertions within attP40 can exhibit decreased msp-300 transcript levels in third instar larvae. Finally, chromosomes carrying certain “transgenic RNAi project” (TRiP) insertions into attP40 can confer pupal or adult inviability or infertility, or dominant nuclear clustering effects in certain genetic backgrounds. These phenotypes do not require transcription from the insertions within attP40. These results demonstrate that attP40 and insertion derivatives act as msp-300 insertional mutations. These findings should be considered when interpreting data from attP40-bearing flies.Item The Drosophila Nesprin-1 homolog MSP300 is required for muscle autophagy and proteostasis(The Company of Biologists, 2024) van der Graaf, Kevin; Srivastav, Saurabh; Nishad, Rajkishor; Stern, Michael; McNew, James A.Nesprin proteins, which are components of the linker of nucleoskeleton and cytoskeleton (LINC) complex, are located within the nuclear envelope and play prominent roles in nuclear architecture. For example, LINC complex proteins interact with both chromatin and the cytoskeleton. Here, we report that the Drosophila Nesprin MSP300 has an additional function in autophagy within larval body wall muscles. RNAi-mediated MSP300 knockdown in larval body wall muscles resulted in defects in the contractile apparatus, muscle degeneration and defective autophagy. In particular, MSP300 knockdown caused accumulation of cytoplasmic aggregates that contained poly-ubiquitylated cargo, as well as the autophagy receptor ref(2)P (the fly homolog of p62 or SQSTM) and Atg8a. Furthermore, MSP300 knockdown larvae expressing an mCherry–GFP-tagged Atg8a transgene exhibited aberrant persistence of the GFP signal within these aggregates, indicating failure of autophagosome maturation. These autophagy deficits were similar to those exhibited by loss of the endoplasmic reticulum (ER) fusion protein Atlastin (Atl), raising the possibility that Atl and MSP300 might function in the same pathway. In support of this possibility, we found that a GFP-tagged MSP300 protein trap exhibited extensive localization to the ER. Alteration of ER-directed MSP300 might abrogate important cytoskeletal contacts necessary for autophagosome completion.