scholarly journals Congenital disorder of glycosylation caused by starting site-specific variant in syntaxin-5

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Peter T. A. Linders ◽  
Eveline C. F. Gerretsen ◽  
Angel Ashikov ◽  
Mari-Anne Vals ◽  
Rinse de Boer ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dermal Fibroblasts ◽  
Protein Isoforms ◽  
Receptor Protein ◽  
Multisystem Disease ◽  
Golgi Transport ◽  
Protein Receptor ◽  
Severe Liver Disease

AbstractThe SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identify a human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Primary human dermal fibroblasts isolated from these patients show defective glycosylation, altered Golgi morphology as measured by electron microscopy, mislocalization of glycosyltransferases, and compromised ER-Golgi trafficking. Measurements of cognate binding SNAREs, based on biotin-synchronizable forms of Stx5 (the RUSH system) and Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. Alternative starting codons of Stx5 are thus linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking.

scholarly journals Congenital disorder of glycosylation caused by starting site-specific variant in syntaxin-5

2020 ◽  
Author(s):  
Peter T.A. Linders ◽  
Eveline C.F. Gerretsen ◽  
Angel Ashikov ◽  
Mari-Anne Vals ◽  
Natalia H. Revelo ◽  
...  
Keyword(s):  
Protein Trafficking ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Dermal Fibroblasts ◽  
Protein Isoforms ◽  
Receptor Protein ◽  
Multisystem Disease ◽  
Golgi Transport ◽  
Protein Receptor ◽  
Severe Liver Disease

AbstractThe SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) protein syntaxin-5 (Stx5) is essential for Golgi transport. In humans, the STX5 mRNA encodes two protein isoforms, Stx5 Long (Stx5L) from the first starting methionine and Stx5 Short (Stx5S) from an alternative starting methionine at position 55. In this study, we identified a novel human disorder caused by a single missense substitution in the second starting methionine (p.M55V), resulting in complete loss of the short isoform. Patients suffer from an early fatal multisystem disease, including severe liver disease, skeletal abnormalities and abnormal glycosylation. Whereas Golgi morphology was unaltered, primary human dermal fibroblasts isolated from these patients showed defective glycosylation and mislocalization of glycosyltransferases. Measurements of anterograde trafficking, based on biotin-synchronizable forms of Stx5 (the RUSH system), and of cognate binding SNAREs, based on Förster resonance energy transfer (FRET), revealed that the short isoform of Stx5 is essential for intra-Golgi transport. This is the first time a mutation in an alternative starting codon is linked to human disease, demonstrating that the site of translation initiation is an important new layer of regulating protein trafficking.

scholarly journals Measuring ligand-dependent and ligand-independent interactions between nuclear receptors and associated proteins using Bioluminescence Resonance Energy Transfer (BRET2)

2006 ◽  
Vol 4 (1) ◽  
pp. nrs.04021 ◽  
Author(s):  
Kristen L. Koterba ◽  
Brian G. Rowan
Keyword(s):  
Energy Transfer ◽  
Protein Interactions ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Renilla Luciferase ◽  
Receptor Protein ◽  
Protein Protein Interactions

Bioluminescent resonance energy transfer (BRET2) is a recently developed technology for the measurement of protein-protein interactions in a live, cell-based system. BRET2 is characterized by the efficient transfer of excited energy between a bioluminescent donor molecule (Renilla luciferase) and a fluorescent acceptor molecule (a mutant of Green Fluorescent Protein (GFP2)). The BRET2 assay offers advantages over fluorescence resonance energy transfer (FRET) because it does not require an external light source thereby eliminating problems of photobleaching and autoflourescence. The absence of contamination by light results in low background that permits detection of very small changes in the BRET2 signal. BRET2 is dependent on the orientation and distance between two fusion proteins and therefore requires extensive preliminary standardization experiments to conclude a positive BRET2 signal independent of variations in protein titrations and arrangement in tertiary structures. Estrogen receptor (ER) signaling is modulated by steroid receptor coactivator 1 (SRC-1). To establish BRET2 in a ligand inducible system we used SRC-1 as the donor moiety and ER as the acceptor moiety. Expression and functionality of the fusion proteins were assessed by transient transfection in HEK-293 cells followed by Western blot analysis and measurement of ER-dependent reporter gene activity. These preliminary determinations are required prior to measuring nuclear receptor protein-protein interactions by BRET2. This article describes in detail the BRET2 methodology for measuring interaction between full-length ER and coregulator proteins in real-time, in an in vivo environment.

scholarly journals GPCR-dependent biasing of GIRK channel signaling dynamics by RGS6 in mouse sinoatrial nodal cells

2020 ◽  
Vol 117 (25) ◽  
pp. 14522-14531
Author(s):  
Allison Anderson ◽  
Ikuo Masuho ◽  
Ezequiel Marron Fernandez de Velasco ◽  
Atsushi Nakano ◽  
Lutz Birnbaumer ◽  
...  
Keyword(s):  
G Protein ◽  
Coupling Efficiency ◽  
Resonance Energy Transfer ◽  
Cardiac Pacemaker ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Protein Isoforms ◽  
Minimal Impact ◽  
Signaling Dynamics ◽  
The Impact

How G protein-coupled receptors (GPCRs) evoke specific biological outcomes while utilizing a limited array of G proteins and effectors is poorly understood, particularly in native cell systems. Here, we examined signaling evoked by muscarinic (M2R) and adenosine (A1R) receptor activation in the mouse sinoatrial node (SAN), the cardiac pacemaker. M2R and A1R activate a shared pool of cardiac G protein-gated inwardly rectifying K+(GIRK) channels in SAN cells from adult mice, but A1R-GIRK responses are smaller and slower than M2R-GIRK responses. Recordings from mice lacking Regulator of G protein Signaling 6 (RGS6) revealed that RGS6 exerts a GPCR-dependent influence on GIRK-dependent signaling in SAN cells, suppressing M2R-GIRK coupling efficiency and kinetics and A1R-GIRK signaling amplitude. Fast kinetic bioluminescence resonance energy transfer assays in transfected HEK cells showed that RGS6 prefers Gαoover Gαias a substrate for its catalytic activity and that M2R signals preferentially via Gαo, while A1R does not discriminate between inhibitory G protein isoforms. The impact of atrial/SAN-selective ablation of Gαoor Gαi2was consistent with these findings. Gαi2ablation had minimal impact on M2R-GIRK and A1R-GIRK signaling in SAN cells. In contrast, Gαoablation decreased the amplitude and slowed the kinetics of M2R-GIRK responses, while enhancing the sensitivity and prolonging the deactivation rate of A1R-GIRK signaling. Collectively, our data show that differences in GPCR-G protein coupling preferences, and the Gαosubstrate preference of RGS6, shape A1R- and M2R-GIRK signaling dynamics in mouse SAN cells.

scholarly journals A High-Throughput Screen for Receptor Protein Tyrosine Phosphatase–γ Selective Inhibitors

2011 ◽  
Vol 16 (5) ◽  
pp. 476-485 ◽  
Author(s):  
Kingsley K. Appiah ◽  
Walter A. Kostich ◽  
Samuel W. Gerritz ◽  
Yanling Huang ◽  
Brian D. Hamman ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Protein ◽  
Protein Tyrosine ◽  
Compound Collection ◽  
Ptp 1B

Protein tyrosine phosphatase–γ (PTP-γ) is a receptor-like PTP whose biological function is poorly understood. A recent mouse PTP-γ genetic deletion model associated the loss of PTP-γ gene expression with a potential antidepressant phenotype. This led the authors to screen a subset of the Bristol-Myers Squibb (BMS) compound collection to identify selective small-molecule inhibitors of receptor-like PTP-γ (RPTP-γ) for use in evaluating enzyme function in vivo. Here, they report the design of a high-throughput fluorescence resonance energy transfer (FRET) assay based on the Z′-LYTE technology to screen for inhibitors of RPTP-γ. A subset of the BMS diverse compound collection was screened and several compounds identified as RPTP-γ inhibitors in the assay. After chemical triage and clustering, compounds were assessed for potency and selectivity by IC50 determination with RPTP-γ and two other phosphatases, PTP-1B and CD45. One hundred twenty-nine RPTP-γ selective (defined as IC50 value greater than 5- to 10-fold over PTP-1B and CD45) inhibitors were identified and prioritized for evaluation. One of these hits, 3-(3, 4-dichlorobenzylthio) thiophene-2-carboxylic acid, was the primary chemotype for the initiation of a medicinal chemistry program.

scholarly journals SNAP-23 regulates phagosome formation and maturation in macrophages

2012 ◽  
Vol 23 (24) ◽  
pp. 4849-4863 ◽  
Author(s):  
Chiye Sakurai ◽  
Hitoshi Hashimoto ◽  
Hideki Nakanishi ◽  
Seisuke Arai ◽  
Yoh Wada ◽  
...  
Keyword(s):  
Critical Role ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Nadph Oxidase Complex ◽  
Uptake Activity ◽  
Fret Efficiency ◽  
Precise Role

Synaptosomal associated protein of 23 kDa (SNAP-23), a plasma membrane–localized soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE), has been implicated in phagocytosis by macrophages. For elucidation of its precise role in this process, a macrophage line overexpressing monomeric Venus–tagged SNAP-23 was established. These cells showed enhanced Fc receptor–mediated phagocytosis. Detailed analyses of each process of phagocytosis revealed a marked increase in the production of reactive oxygen species within phagosomes. Also, enhanced accumulation of a lysotropic dye, as well as augmented quenching of a pH-sensitive fluorophore were observed. Analyses of isolated phagosomes indicated the critical role of SNAP-23 in the functional recruitment of the NADPH oxidase complex and vacuolar-type H+-ATPase to phagosomes. The data from the overexpression experiments were confirmed by SNAP-23 knockdown, which demonstrated a significant delay in phagosome maturation and a reduction in uptake activity. Finally, for analyzing whether phagosomal SNAP-23 entails a structural change in the protein, an intramolecular Förster resonance energy transfer (FRET) probe was constructed, in which the distance within a TagGFP2-TagRFP was altered upon close approximation of the N-termini of its two SNARE motifs. FRET efficiency on phagosomes was markedly enhanced only when VAMP7, a lysosomal SNARE, was coexpressed. Taken together, our results strongly suggest the involvement of SNAP-23 in both phagosome formation and maturation in macrophages, presumably by mediating SNARE-based membrane traffic.

scholarly journals Agonist-induced formation of unproductive receptor-G12complexes

2020 ◽  
Vol 117 (35) ◽  
pp. 21723-21730
Author(s):  
Najeah Okashah ◽  
Shane C. Wright ◽  
Kouki Kawakami ◽  
Signe Mathiasen ◽  
Joris Zhou ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Receptor Activation ◽  
Receptor Internalization ◽  
Protein Association ◽  
Protein Activation ◽  
Protein Receptor ◽  
G Protein Activation

G proteins are activated when they associate with G protein-coupled receptors (GPCRs), often in response to agonist-mediated receptor activation. It is generally thought that agonist-induced receptor-G protein association necessarily promotes G protein activation and, conversely, that activated GPCRs do not interact with G proteins that they do not activate. Here we show that GPCRs can form agonist-dependent complexes with G proteins that they do not activate. Using cell-based bioluminescence resonance energy transfer (BRET) and luminescence assays we find that vasopressin V2receptors (V2R) associate with both Gsand G12heterotrimers when stimulated with the agonist arginine vasopressin (AVP). However, unlike V2R-Gscomplexes, V2R-G12complexes are not destabilized by guanine nucleotides and do not promote G12activation. Activating V2R does not lead to signaling responses downstream of G12activation, but instead inhibits basal G12-mediated signaling, presumably by sequestering G12heterotrimers. Overexpressing G12inhibits G protein receptor kinase (GRK) and arrestin recruitment to V2R and receptor internalization. Formyl peptide (FPR1 and FPR2) and Smoothened (Smo) receptors also form complexes with G12that are insensitive to nucleotides, suggesting that unproductive GPCR-G12complexes are not unique to V2R. These results indicate that agonist-dependent receptor-G protein association does not always lead to G protein activation and may in fact inhibit G protein activation.

scholarly journals Syntaxin 11 regulates the stimulus-dependent transport of Toll-like receptor 4 to the plasma membrane by cooperating with SNAP-23 in macrophages

2019 ◽  
Vol 30 (9) ◽  
pp. 1085-1097 ◽  
Author(s):  
Daiki Kinoshita ◽  
Chiye Sakurai ◽  
Maya Morita ◽  
Masashi Tsunematsu ◽  
Naohiro Hori ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Transport ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Interferon Γ ◽  
Toll Like Receptor ◽  
Toll Like Receptor 4 ◽  
Protein Receptor ◽  
Syntaxin 11 ◽  
Dependent Transport

Syntaxin 11 (stx11) is a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) that is selectively expressed in immune cells; however, its precise role in macrophages is unclear. We showed that stx11 knockdown reduces the phagocytosis of Escherichia coli in interferon-γ–activated macrophages. stx11 knockdown decreased Toll-like receptor 4 (TLR4) localization on the plasma membrane without affecting total expression. Plasma membrane–localized TLR4 was primarily endocytosed within 1 h by lipopolysaccharide (LPS) stimulation and gradually relocalized 4 h after removal of LPS. This relocalization was significantly impaired by stx11 knockdown. The lack of TLR4 transport to the plasma membrane is presumably related to TLR4 degradation in acidic endosomal organelles. Additionally, an immunoprecipitation experiment suggested that stx11 interacts with SNAP-23, a plasma membrane–localized SNARE protein, whose depletion also inhibits TLR4 replenishment in LPS-stimulated cells. Using an intramolecular Förster resonance energy transfer (FRET) probe for SNAP-23, we showed that the high FRET efficiency caused by LPS stimulation is reduced by stx11 knockdown. These findings suggest that stx11 regulates the stimulus-dependent transport of TLR4 to the plasma membrane by cooperating with SNAP-23 in macrophages. Our results clarify the regulatory mechanisms underlying intracellular transport of TLR4 and have implications for microbial pathogenesis and immune responses.

scholarly journals Oligomeric Architecture of Mouse Activating Nkrp1 Receptors on Living Cells

2019 ◽  
Vol 20 (8) ◽  
pp. 1884 ◽  
Author(s):  
Ljubina Adámková ◽  
Zuzana Kvíčalová ◽  
Daniel Rozbeský ◽  
Zdeněk Kukačka ◽  
David Adámek ◽  
...  
Keyword(s):  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Living Cells ◽  
Lymphoid Cells ◽  
Protein Isoforms ◽  
Major Influence ◽  
Protein Dimerization ◽  
Transmembrane Receptors ◽  
Insight Into

Mouse activating Nkrp1 proteins are commonly described as type II transmembrane receptors with disulfide-linked homodimeric structure. Their function and the manner in which Nkrp1 proteins of mouse strain (C57BL/6) oligomerize are still poorly understood. To assess the oligomerization state of Nkrp1 proteins, mouse activating EGFP-Nkrp1s were expressed in mammalian lymphoid cells and their oligomerization evaluated by Förster resonance energy transfer (FRET). Alternatively, Nkrp1s oligomers were detected by Western blotting to specify the ratio between monomeric and dimeric forms. We also performed structural characterization of recombinant ectodomains of activating Nkrp1 receptors. Nkrp1 isoforms c1, c2 and f were expressed prevalently as homodimers, whereas the Nkrp1a displays larger proportion of monomers on the cell surface. Cysteine-to-serine mutants revealed the importance of all stalk cysteines for protein dimerization in living cells with a major influence of cysteine at position 74 in two Nkrp1 protein isoforms. Our results represent a new insight into the oligomerization of Nkrp1 receptors on lymphoid cells, which will help to determine their function.

scholarly journals Ubiquilin 1 Modulates Amyloid Precursor Protein Trafficking and Aβ Secretion

2006 ◽  
Vol 281 (43) ◽  
pp. 32240-32253 ◽  
Author(s):  
Mikko Hiltunen ◽  
Alice Lu ◽  
Anne V. Thomas ◽  
Donna M. Romano ◽  
Minji Kim ◽  
...  
Keyword(s):  
Cell Surface ◽  
Amyloid Precursor Protein ◽  
Protein Trafficking ◽  
Resonance Energy Transfer ◽  
Neuronal Cell ◽  
Resonance Energy ◽  
Precursor Protein ◽  
Intact Cells ◽  
App Trafficking ◽  
Intracellular Compartments

Ubiquilin 1 (UBQLN1) is a ubiquitin-like protein, which has been shown to play a central role in regulating the proteasomal degradation of various proteins, including the presenilins. We recently reported that DNA variants in UBQLN1 increase the risk for Alzheimer disease, by influencing expression of this gene in brain. Here we present the first assessment of the effects of UBQLN1 on the metabolism of the amyloid precursor protein (APP). For this purpose, we employed RNA interference to down-regulate UBQLN1 in a variety of neuronal and non-neuronal cell lines. We demonstrate that down-regulation of UBQLN1 accelerates the maturation and intracellular trafficking of APP, while not interfering with α-, β-, or γ-secretase levels or activity. UBQLN1 knockdown increased the ratio of APP mature/immature, increased levels of full-length APP on the cell surface, and enhanced the secretion of sAPP (α- and β-forms). Moreover, UBQLN1 knockdown increased levels of secreted Aβ40 and Aβ42. Finally, employing a fluorescence resonance energy transfer-based assay, we show that UBQLN1 and APP come into close proximity in intact cells, independently of the presence of the presenilins. Collectively, our findings suggest that UBQLN1 may normally serve as a cytoplasmic “gatekeeper” that may control APP trafficking from intracellular compartments to the cell surface. These findings suggest that changes in UBQLN1 steady-state levels affect APP trafficking and processing, thereby influencing the generation of Aβ.

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