Fluorescent TAP as a Platform for Virus-Induced Degradation of the Antigenic Peptide Transporter

Transporter associated with antigen processing (TAP), a key player in the major histocompatibility complex class I-restricted antigen presentation, makes an attractive target for viruses that aim to escape the immune system. Mechanisms of TAP inhibition vary among virus species. Bovine herpesvirus 1 (BoHV-1) is unique in its ability to target TAP for proteasomal degradation following conformational arrest by the UL49.5 gene product. The exact mechanism of TAP removal still requires elucidation. For this purpose, a TAP-GFP (green fluorescent protein) fusion protein is instrumental, yet GFP-tagging may affect UL49.5-induced degradation. Therefore, we constructed a series of TAP-GFP variants using various linkers to obtain an optimal cellular fluorescent TAP platform. Mel JuSo (MJS) cells with CRISPR/Cas9 TAP1 or TAP2 knockouts were reconstituted with TAP-GFP constructs. Our results point towards a critical role of GFP localization on fluorescent properties of the fusion proteins and, in concert with the type of a linker, on the susceptibility to virally-induced inhibition and degradation. The fluorescent TAP platform was also used to re-evaluate TAP stability in the presence of other known viral TAP inhibitors, among which only UL49.5 was able to reduce TAP levels. Finally, we provide evidence that BoHV-1 UL49.5-induced TAP removal is p97-dependent, which indicates its degradation via endoplasmic reticulum-associated degradation (ERAD).

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Rapid removal of phagosomal ferroportin in macrophages contributes to nutritional immunity

Blood Advances ◽

10.1182/bloodadvances.2020002833 ◽

2021 ◽

Vol 5 (2) ◽

pp. 459-474

Author(s):

Ronald S. Flannagan ◽

Tayler J. Farrell ◽

Steven M. Trothen ◽

Jimmy D. Dikeakos ◽

David E. Heinrichs

Keyword(s):

Fluorescent Protein ◽

Critical Role ◽

Cellular Level ◽

Protein Fusion ◽

Extracellular Milieu ◽

Nutritional Immunity ◽

Protein Receptor ◽

Serovar Typhimurium ◽

Green Fluorescent ◽

Phagosomal Membrane

Abstract Nutrient sequestration is an essential facet of host innate immunity. Macrophages play a critical role in controlling iron availability through expression of the iron transport protein ferroportin (FPN), which extrudes iron from the cytoplasm to the extracellular milieu. During phagocytosis, the limiting phagosomal membrane, which derives from the plasmalemma, can be decorated with FPN and, if functional, will move iron from the cytosol into the phagosome lumen. This serves to feed iron to phagocytosed microbes and would be counterproductive to the many other known host mechanisms working to starve microbes of this essential metal. To understand how FPN is regulated during phagocytosis, we expressed FPN as a green fluorescent protein–fusion protein in macrophages and monitored its localization during uptake of various phagocytic targets, including Staphylococcus aureus, Salmonella enterica serovar Typhimurium, human erythrocytes, and immunoglobulin G opsonized latex beads. We find that FPN is rapidly removed, independently of Vps34 and PI(3)P, from early phagosomes and does not follow recycling pathways that regulate transferrin receptor recycling. Live-cell video microscopy showed that FPN movement on the phagosome is dynamic, with punctate and tubular structures forming before FPN is trafficked back to the plasmalemma. N-ethylmaleimide–sensitive factor, which disrupts soluble NSF attachment protein receptor (SNARE)–mediated membrane fusion and trafficking, prevented FPN removal from the phagosome. Our data support the hypothesis that removal of FPN from the limiting phagosomal membrane will, at the cellular level, ensure that iron cannot be pumped into phagosomes. We propose this as yet another mechanism of host nutritional immunity to subvert microbial growth.

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Co-operative function and mutual stabilization of the half ATP-binding cassette transporters HAF-4 and HAF-9 in Caenorhabditis elegans

Biochemical Journal ◽

10.1042/bj20130115 ◽

2013 ◽

Vol 452 (3) ◽

pp. 467-475 ◽

Cited By ~ 7

Author(s):

Takahiro Tanji ◽

Kenji Nishikori ◽

Hirohisa Shiraishi ◽

Masatomo Maeda ◽

Ayako Ohashi-Kobayashi

Keyword(s):

Caenorhabditis Elegans ◽

Antigen Processing ◽

Fluorescent Protein ◽

Peptide Transporter ◽

Deletion Mutants ◽

Atp Binding ◽

Physical Interaction ◽

Atp Binding Cassette Transporters ◽

Green Fluorescent ◽

Atp Binding Cassette

Caenorhabditis elegans HAF-4 and HAF-9 are half ABC (ATP-binding-cassette) transporters that are highly homologous to the human lysosomal peptide transporter TAPL [TAP (transporter associated with antigen processing)-like; ABCB9]. We reported previously that both HAF-4 and HAF-9 localize to the membrane of a subset of intestinal organelles, and are required for the formation of these organelles and other physiological aspects. In the present paper, we report the genetic and physical interactions between HAF-4 and HAF-9. Overexpression of HAF-4 and HAF-9 did not rescue the intestinal organelle defect of the haf-9 and haf-4 deletion mutants respectively, indicating that they cannot substitute for each other. Double haf-4 and haf-9 mutants do not exhibit more severe phenotypes than the single mutants, suggesting their co-operative function. Immunoprecipitation experiments demonstrated their physical interaction. The results of the present study suggest that HAF-4 and HAF-9 form a heterodimer. Furthermore, Western blot analysis of the deletion mutants and RNAi (RNA interference) knockdown experiments in GFP (green fluorescent protein)-tagged HAF-4 or HAF-9 transgenic worms suggest that HAF-4–HAF-9 heterodimer formation is required for their stabilization. The findings provide a clue as to how ABC transporters adopt a stable functional form.

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Potassium Uptake Modulates Staphylococcus aureus Metabolism

mSphere ◽

10.1128/msphere.00125-16 ◽

2016 ◽

Vol 1 (3) ◽

Cited By ~ 7

Author(s):

Casey M. Gries ◽

Marat R. Sadykov ◽

Logan L. Bulock ◽

Sujata S. Chaudhari ◽

Vinai C. Thomas ◽

...

Keyword(s):

Carbon Flux ◽

Fluorescent Protein ◽

Critical Role ◽

Normal Growth ◽

Cytoplasmic Ph ◽

Carbon Utilization ◽

Content Type ◽

Alkaline Conditions ◽

Green Fluorescent

ABSTRACT Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization. As a leading cause of community-associated and nosocomial infections, Staphylococcus aureus requires sophisticated mechanisms that function to maintain cellular homeostasis in response to its exposure to changing environmental conditions. The adaptation to stress and maintenance of homeostasis depend largely on membrane activity, including supporting electrochemical gradients and synthesis of ATP. This is largely achieved through potassium (K+) transport, which plays an essential role in maintaining chemiosmotic homeostasis, affects antimicrobial resistance, and contributes to fitness in vivo. Here, we report that S. aureus Ktr-mediated K+ uptake is necessary for maintaining cytoplasmic pH and the establishment of a proton motive force. Metabolite analyses revealed that K+ deficiency affects both metabolic and energy states of S. aureus by impairing oxidative phosphorylation and directing carbon flux toward substrate-level phosphorylation. Taken together, these results underline the importance of K+ uptake in maintaining essential components of S. aureus metabolism. IMPORTANCE Previous studies describing mechanisms for K+ uptake in S. aureus revealed that the Ktr-mediated K+ transport system was required for normal growth under alkaline conditions but not under neutral or acidic conditions. This work focuses on the effect of K+ uptake on S. aureus metabolism, including intracellular pH and carbon flux, and is the first to utilize a pH-dependent green fluorescent protein (GFP) to measure S. aureus cytoplasmic pH. These studies highlight the role of K+ uptake in supporting proton efflux under alkaline conditions and uncover a critical role for K+ uptake in establishing efficient carbon utilization.

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Role of Septins and the Exocyst Complex in the Function of Hydrolytic Enzymes Responsible for Fission Yeast Cell Separation

Molecular Biology of the Cell ◽

10.1091/mbc.e04-12-1114 ◽

2005 ◽

Vol 16 (10) ◽

pp. 4867-4881 ◽

Cited By ~ 57

Author(s):

Ana Belén Martín-Cuadrado ◽

Jennifer L. Morrell ◽

Mami Konomi ◽

Hanbing An ◽

Claudia Petit ◽

...

Keyword(s):

Cell Separation ◽

Fluorescent Protein ◽

Hydrolytic Enzymes ◽

Protein Fusion ◽

Septin Ring ◽

Cargo Proteins ◽

Positional Marker ◽

A Cell ◽

Green Fluorescent

Cell separation in Schizosaccharomyces pombe is achieved by the concerted action of the Eng1 endo-β-1,3-glucanase and the Agn1 endo-α-1,3-glucanase, which are transported to the septum and localize to a ringlike structure that surrounds the septum. The requirements for the correct localization of both hydrolases as a ring were analyzed using green fluorescent protein fusion proteins. Targeting to the septum required a functional exocyst, because both proteins failed to localize correctly in sec8-1 or exo70Δ mutants, suggesting that Agn1 and Eng1 might be two of the cargo proteins present in the vesicles that accumulate in exocyst mutants. Septins and Mid2 were also required for correct formation of a ring. In their absence, Eng1 and Agn1 were found in a disklike structure that spanned the septum, rather than in a ring. Even though septin and mid2Δ mutants have a cell separation defect, the septum and the distribution of linear β-1,3-glucans were normal in these cells, suggesting that mislocalization of Eng1 and Agn1 might be the reason underlying the failure to separate efficiently. Thus, one of the functions of the septin ring would be to act as a positional marker for the localization of hydrolytic proteins to the medial region.

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Mutations in Conserved Regions of the Predicted RAG2 Kelch Repeats Block Initiation of V(D)J Recombination and Result in Primary Immunodeficiencies

Molecular and Cellular Biology ◽

10.1128/mcb.20.15.5653-5664.2000 ◽

2000 ◽

Vol 20 (15) ◽

pp. 5653-5664 ◽

Cited By ~ 46

Author(s):

Carlos A. Gomez ◽

Leon M. Ptaszek ◽

Anna Villa ◽

Fabio Bozzi ◽

Cristina Sobacchi ◽

...

Keyword(s):

Fluorescent Protein ◽

Critical Role ◽

Recombination Reaction ◽

Signal Recognition ◽

Mutant Proteins ◽

Recombination Signal ◽

Loop Regions ◽

Green Fluorescent ◽

Processing Steps

ABSTRACT The V(D)J recombination reaction is composed of multiple nucleolytic processing steps mediated by the recombination-activating proteins RAG1 and RAG2. Sequence analysis has suggested that RAG2 contains six kelch repeat motifs that are predicted to form a six-bladed β-propeller structure, with the second β-strand of each repeat demonstrating marked conservation both within and between kelch repeat-containing proteins. Here we demonstrate that mutations G95R and ΔI273 within the predicted second β-strand of repeats 2 and 5 of RAG2 lead to immunodeficiency in patients P1 and P2. Green fluorescent protein fusions with the mutant proteins reveal appropriate localization to the nucleus. However, both mutations reduce the capacity of RAG2 to interact with RAG1 and block recombination signal cleavage, therefore implicating a defect in the early steps of the recombination reaction as the basis of the clinical phenotype. The present experiments, performed with an extensive panel of site-directed mutations within each of the six kelch motifs, further support the critical role of both hydrophobic and glycine-rich regions within the second β-strand for RAG1-RAG2 interaction and recombination signal recognition and cleavage. In contrast, multiple mutations within the variable-loop regions of the kelch repeats had either mild or no effects on RAG1-RAG2 interaction and hence on the ability to mediate recombination. In all, the data demonstrate a critical role of the RAG2 kelch repeats for V(D)J recombination and highlight the importance of the conserved elements of the kelch motif.

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Critical role of CFTR-dependent lipid rafts in cigarette smoke-induced lung epithelial injury

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00408.2010 ◽

2011 ◽

Vol 300 (6) ◽

pp. L811-L820 ◽

Cited By ~ 64

Author(s):

Manish Bodas ◽

Taehong Min ◽

Neeraj Vij

Keyword(s):

Cigarette Smoke ◽

Lipid Raft ◽

Fluorescent Protein ◽

Critical Role ◽

Zonula Occludens ◽

Lung Epithelial ◽

Underlying Mechanisms ◽

Green Fluorescent ◽

Ceramide Accumulation

Apoptosis of lung epithelial and endothelial cells by exposure to cigarette smoke (CS) severely damages the lung tissue, leading to the pathogenesis of emphysema, but the underlying mechanisms are poorly understood. We have recently established a direct correlation between decreased lipid raft CFTR expression and emphysema progression through increased ceramide accumulation. In the present work, we investigated the role of membrane CFTR in regulating apoptosis and autophagy responses to CS exposure. We report a constitutive and CS-induced increase in the number of TUNEL-positive apoptotic cells in Cftr−/− murine lungs compared with Cftr+/+ murine lungs that also correlated with a concurrent increase in the expression of ceramide, NF-κB, CD95/Fas, lipid raft proteins, and zonula occludens (ZO)-1/2 ( P < 0.001). We also verified that stable wild-type CFTR expression in CFBE41o− cells controls constitutively elevated caspase-3/7 activity (−1.6-fold, P < 0.001). Our data suggest that membrane CFTR regulates ceramide-enriched lipid raft signaling platforms required for the induction of Fas-mediated apoptotic signaling. In addition, lack of membrane CFTR also modulates autophagy, as demonstrated by the significant increase in constitutive ( P < 0.001) and CSE-induced ( P < 0.005) perinuclear accumulation of green fluorescent protein-microtubule-associated protein 1 light chain-3 (LC3) in the absence of membrane CFTR (CFBE41o− cells). The significant constitutive and CS-induced increase ( P < 0.05) in p62 and LC3β expression in CFTR-deficient cells and mice corroborates these findings and suggest a defective autophagy response in the absence of membrane CFTR. Our data demonstrate the critical role of membrane-localized CFTR in regulating apoptotic and autophagic responses in CS-induced lung injury that may be involved in the pathogenesis of severe emphysema.

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The Role of the Protein Matrix in Green Fluorescent Protein Fluorescence

Photochemistry and Photobiology ◽

10.1562/2005-04-11-ra-485 ◽

2006 ◽

Vol 82 (2) ◽

pp. 367 ◽

Cited By ~ 52

Author(s):

Scott L. Maddalo ◽

Marc Zimmer

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Green Fluorescent Protein Fluorescence ◽

Protein Fluorescence ◽

Protein Matrix ◽

Green Fluorescent

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20-HETE-mediated cytotoxicity and apoptosis in ischemic kidney epithelial cells

AJP Renal Physiology ◽

10.1152/ajprenal.00387.2007 ◽

2008 ◽

Vol 294 (3) ◽

pp. F562-F570 ◽

Cited By ~ 37

Author(s):

Vani Nilakantan ◽

Cheryl Maenpaa ◽

Guangfu Jia ◽

Richard J. Roman ◽

Frank Park

Keyword(s):

Caspase 3 ◽

Fluorescent Protein ◽

Ischemia Reperfusion ◽

Atp Depletion ◽

Cellular Damage ◽

Apoptotic Pathway ◽

Injury Model ◽

Green Fluorescent

20-HETE, a metabolite of arachidonic acid, has been implicated as a mediator of free radical formation and tissue death following ischemia-reperfusion (IR) injury in the brain and heart. The present study examined the role of this pathway in a simulated IR renal injury model in vitro. Modified self-inactivating lentiviral vectors were generated to stably overexpress murine Cyp4a12 following transduction into LLC-PK1 cells (LLC-Cyp4a12). We compared the survival of control and transduced LLC-PK1 cells following 4 h of ATP depletion and 2 h of recovery in serum-free medium. ATP depletion-recovery of LLC-Cyp4a12 cells resulted in a significantly higher LDH release ( P < 0.05) compared with LLC-enhanced green fluorescent protein (EGFP) cells. Treatment with the SOD mimetic MnTMPyP (100 μM) resulted in decreased cytotoxicity in LLC-Cyp4a12 cells. The selective 20-HETE inhibitor HET-0016 (10 μM) also inhibited cytotoxicity significantly ( P < 0.05) in LLC-Cyp4a12 cells. Dihydroethidium fluorescence showed that superoxide levels were increased to the same degree in LLC-EGFP and LLC-Cyp4a12 cells after ATP depletion-recovery compared with control cells and that this increase was inhibited by MnTMPyP. There was a significant increase ( P < 0.05) of caspase-3 cleavage, an effector protease of the apoptotic pathway, in the LLC-Cyp4a12 vs. LLC-EGFP cells ( P < 0.05). This was abolished in the presence of HET-0016 ( P < 0.05) or MnTMPyP ( P < 0.01). These results demonstrate that 20-HETE overexpression can significantly exacerbate the cellular damage that is associated with renal IR injury and that the programmed cell death is mediated by activation of caspase-3 and is partially dependent on enhanced CYP4A generation of free radicals.

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