Probing Protein Distribution Along the Nuclear Envelope In Vivo by Using Single-Point FRAP

AbstractMitochondrial complex I is central to the pathological reactive oxygen species (ROS) production that underlies cardiac ischemia–reperfusion (IR) injury. ND6-P25L mice are homoplasmic for a disease-causing mtDNA point mutation encoding the P25L substitution in the ND6 subunit of complex I. The cryo-EM structure of ND6-P25L complex I revealed subtle structural changes that facilitate rapid conversion to the “deactive” state, usually formed only after prolonged inactivity. Despite its tendency to adopt the “deactive” state, the mutant complex is fully active for NADH oxidation, but cannot generate ROS by reverse electron transfer (RET). ND6-P25L mitochondria function normally, except for their lack of RET ROS production, and ND6-P25L mice are protected against cardiac IR injury in vivo. Thus, this single point mutation in complex I, which does not affect oxidative phosphorylation but renders the complex unable to catalyse RET, demonstrates the pathological role of ROS production by RET during IR injury.

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In Vivo Evidence for TonB Dimerization

Journal of Bacteriology ◽

10.1128/jb.185.19.5747-5754.2003 ◽

2003 ◽

Vol 185 (19) ◽

pp. 5747-5754 ◽

Cited By ~ 50

Author(s):

Annette Sauter ◽

S. Peter Howard ◽

Volkmar Braun

Keyword(s):

Single Point ◽

Point Mutations ◽

Ferric Citrate ◽

Site Directed Mutagenesis ◽

Toxin Gene ◽

Transmembrane Region ◽

Dimer Formation ◽

Citrate Transport ◽

Hybrid Proteins

ABSTRACT TonB, in complex with ExbB and ExbD, is required for the energy-dependent transport of ferric siderophores across the outer membrane of Escherichia coli, the killing of cells by group B colicins, and infection by phages T1 and φ80. To gain insights into the protein complex, TonB dimerization was studied by constructing hybrid proteins from complete TonB (containing amino acids 1 to 239) [TonB(1-239)] and the cytoplasmic fragment of ToxR which, when dimerized, activates the transcription of the cholera toxin gene ctx. ToxR(1-182)-TonB(1-239) activated the transcription of lacZ under the control of the ctx promoter (P ctx ::lacZ). Replacement of the TonB transmembrane region by the ToxR transmembrane region resulted in the hybrid proteins ToxR(1-210)-TonB(33-239) and ToxR(1-210)-TonB(164-239), of which only the latter activated P ctx ::lacZ transcription. Dimer formation was reduced but not abolished in a mutant lacking ExbB and ExbD, suggesting that these complex components may influence dimerization but are not strictly required and that the N-terminal cytoplasmic membrane anchor and the C-terminal region are important for dimer formation. The periplasmic TonB fragment, TonB(33-239), inhibits ferrichrome and ferric citrate transport and induction of the ferric citrate transport system. This competition provided a means to positively screen for TonB(33-239) mutants which displayed no inhibition. Single point mutations of inactive fragments selected in this manner were introduced into complete TonB, and the phenotypes of the TonB mutant strains were determined. The mutations located in the C-terminal half of TonB, three of which (Y163C, V188E, and R204C) were obtained separately by site-directed mutagenesis, as was the isolated F230V mutation, were studied in more detail. They displayed different activity levels for various TonB-dependent functions, suggesting function-related specificities which reflect differences in the interactions of TonB with various transporters and receptors.

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Ribonucleic acid stimulation of mammalian liver nuclear-envelope nucleoside triphosphatase. A possible enzymic marker for the nuclear envelope

Biochemical Journal ◽

10.1042/bj1620671 ◽

1977 ◽

Vol 162 (3) ◽

pp. 671-679 ◽

Cited By ~ 45

Author(s):

P S Agutter ◽

J R Harris ◽

I Stevenson

Keyword(s):

Nuclear Envelope ◽

Specific Activity ◽

Assay Medium ◽

Exogenous Rna ◽

Mammalian Liver ◽

High Concentrations ◽

Enzymic Marker ◽

Stimulation Of ◽

Nucleoside Triphosphatase

1. The specific activity of rat and pig liver nuclear-envelope nucleoside triphosphatase (EC 3.6.1.3) decreases when the system is depleted of RNA. The activity can be restored by adding high concentrations of yeast RNA to the assay medium. 2. Exogenous RNA also increases the activity of the enzyme in control envelopes (not RNA-depleted). The effect appears to be largely specific for poly(A) and poly(G); it is not stimulated by rRNA or tRNA preparations, ribonuclease-hydrolysed RNA, AMP, or double- or single-stranded DNA. 3. Inhibitors of the enzyme, in concentrations at which half-maximal inhibition of the enzyme is achieved, do not affect the percentage stimulation of the enzyme by yeast RNA. 4. The simulation is abolished by the inclusion of 150 mM-KCl or -NaCl in the assay medium, but not by increasing the assay pH to 8.5. 5. The results are discussed in the light of the possible role of the nucleoside triphosphatase in vivo in nucleo-cytoplasmic ribonucleoprotein translocation. 6. It is proposed that poly(G)-stimulated Mg2+-activated adenosine triphosphatase activity should be adopted as an enzymic marker for the nuclear envelope.

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BAF is required for emerin assembly into the reforming nuclear envelope

Journal of Cell Science ◽

10.1242/jcs.114.24.4575 ◽

2001 ◽

Vol 114 (24) ◽

pp. 4575-4585 ◽

Cited By ~ 3

Author(s):

Tokuko Haraguchi ◽

Takako Koujin ◽

Miriam Segura-Totten ◽

Kenneth K. Lee ◽

Yosuke Matsuoka ◽

...

Keyword(s):

Nuclear Envelope ◽

Hela Cells ◽

Core Region ◽

Lamin B ◽

The Core ◽

Double Stranded Dna ◽

Recessive Form ◽

Nuclear Envelope Assembly

Mutations in emerin cause the X-linked recessive form of Emery-Dreifuss muscular dystrophy (EDMD). Emerin localizes at the inner membrane of the nuclear envelope (NE) during interphase, and diffuses into the ER when the NE disassembles during mitosis. We analyzed the recruitment of wildtype and mutant GFP-tagged emerin proteins during nuclear envelope assembly in living HeLa cells. During telophase, emerin accumulates briefly at the ‘core’ region of telophase chromosomes, and later distributes over the entire nuclear rim. Barrier-to-autointegration factor (BAF), a protein that binds nonspecifically to double-stranded DNA in vitro, co-localized with emerin at the ‘core’ region of chromosomes during telophase. An emerin mutant defective for binding to BAF in vitro failed to localize at the ‘core’ in vivo, and subsequently failed to localize at the reformed NE. In HeLa cells that expressed BAF mutant G25E, which did not show ‘core’ localization, the endogenous emerin proteins failed to localize at the ‘core’ region during telophase, and did not assemble into the NE during the subsequent interphase. BAF mutant G25E also dominantly dislocalized LAP2β and lamin A from the NE, but had no effect on the localization of lamin B. We conclude that BAF is required for the assembly of emerin and A-type lamins at the reforming NE during telophase, and may mediate their stability in the subsequent interphase.

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The Positioning and Dynamics of Origins of Replication in the Budding Yeast Nucleus

The Journal of Cell Biology ◽

10.1083/jcb.152.2.385 ◽

2001 ◽

Vol 152 (2) ◽

pp. 385-400 ◽

Cited By ~ 115

Author(s):

Patrick Heun ◽

Thierry Laroche ◽

M.K. Raghuraman ◽

Susan M. Gasser

Keyword(s):

Nuclear Envelope ◽

Chromatin Structure ◽

Fluorescent Protein ◽

Time Lapse ◽

Yeast Cells ◽

G1 Phase ◽

Nuclear Pore ◽

Origin Firing ◽

Green Fluorescent

We have analyzed the subnuclear position of early- and late-firing origins of DNA replication in intact yeast cells using fluorescence in situ hybridization and green fluorescent protein (GFP)–tagged chromosomal domains. In both cases, origin position was determined with respect to the nuclear envelope, as identified by nuclear pore staining or a NUP49-GFP fusion protein. We find that in G1 phase nontelomeric late-firing origins are enriched in a zone immediately adjacent to the nuclear envelope, although this localization does not necessarily persist in S phase. In contrast, early firing origins are randomly localized within the nucleus throughout the cell cycle. If a late-firing telomere-proximal origin is excised from its chromosomal context in G1 phase, it remains late-firing but moves rapidly away from the telomere with which it was associated, suggesting that the positioning of yeast chromosomal domains is highly dynamic. This is confirmed by time-lapse microscopy of GFP-tagged origins in vivo. We propose that sequences flanking late-firing origins help target them to the periphery of the G1-phase nucleus, where a modified chromatin structure can be established. The modified chromatin structure, which would in turn retard origin firing, is both autonomous and mobile within the nucleus.

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Pathologic tau conformer ensembles induce dynamic, liquid-liquid phase separation events at the nuclear envelope

BMC Biology ◽

10.1186/s12915-021-01132-y ◽

2021 ◽

Vol 19 (1) ◽

Cited By ~ 1

Author(s):

Sang-Gyun Kang ◽

Zhuang Zhuang Han ◽

Nathalie Daude ◽

Emily McNamara ◽

Serene Wohlgemuth ◽

...

Keyword(s):

Phase Separation ◽

Liquid Phase ◽

Nuclear Envelope ◽

Guanidine Hydrochloride ◽

Liquid Phase Separation ◽

Soluble Form ◽

Stable Cell Line ◽

Cross Sectional ◽

Liquid Liquid Phase Separation

Abstract Background The microtubule-associated protein tau forms aggregates in different neurodegenerative diseases called tauopathies. Prior work has shown that a single P301L mutation in tau gene, MAPT, can promote alternative tau folding pathways that correlate with divergent clinical diagnoses. Using progressive chemical denaturation, some tau preparations from the brain featured complex transitions starting at low concentrations of guanidine hydrochloride (GdnHCl) denaturant, indicating an ensemble of differently folded tau species called conformers. On the other hand, brain samples with abundant, tangle-like pathology had simple GdnHCl unfolding profile resembling the profile of fibrillized recombinant tau and suggesting a unitary conformer composition. In studies here we sought to understand tau conformer progression and potential relationships with condensed liquid states, as well as associated perturbations in cell biological processes. Results As starting material, we used brain samples from P301L transgenic mice containing tau conformer ensembles that unfolded at low GdnHCl concentrations and with signatures resembling brain material from P301L subjects presenting with language or memory problems. We seeded reporter cells expressing a soluble form of 4 microtubule-binding repeat tau fused to GFP or YFP reporter moieties, resulting in redistribution of dispersed fluorescence signals into focal assemblies that could fuse together and move within processes between adjacent cells. Nuclear envelope fluorescent tau signals and small fluorescent inclusions behaved as a demixed liquid phase, indicative of liquid-liquid phase separation (LLPS); these droplets exhibited spherical morphology, fusion events and could recover from photobleaching. Moreover, juxtanuclear tau assemblies were associated with disrupted nuclear transport and reduced cell viability in a stable cell line. Staining for thioflavin S (ThS) became more prevalent as tau-derived inclusions attained cross-sectional area greater than 3 μm2, indicating (i) a bipartite composition, (ii) in vivo progression of tau conformers, and (iii) that a mass threshold applying to demixed condensates may drive liquid-solid transitions. Conclusions Tau conformer ensembles characterized by denaturation at low GdnHCl concentration templated the production of condensed droplets in living cells. These species exhibit dynamic changes and develop in vivo, and the larger ThS-positive assemblies may represent a waystation to arrive at intracellular fibrillar tau inclusions seen in end-stage genetic tauopathies.

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Sulfated non-anticoagulant heparin derivative modifies intracellular hemoglobin, inhibits cell sickling in vitro, and prolongs survival of sickle cell mice under hypoxia

Haematologica ◽

10.3324/haematol.2020.272393 ◽

2021 ◽

Author(s):

Osheiza Abdulmalik ◽

Noureldien H. E. Darwish ◽

Vandhana Muralidharan-Chari ◽

Maii Abu Taleb ◽

Shaker A. Mousa

Keyword(s):

Sickle Cell ◽

Inflammatory Responses ◽

Anticoagulant Activity ◽

Single Point ◽

Bleeding Complications ◽

Single Point Mutation ◽

Repeated Dosing ◽

Heparin Derivative

Sickle cell disease (SCD) is an autosomal recessive genetic disease caused by a single point mutation, resulting in abnormal sickle hemoglobin (HbS). During hypoxia or dehydration, HbS polymerizes to form insoluble aggregates and induces sickling of red blood cells (RBCs). RBC sickling increases adhesiveness of RBCs to alter the rheological properties of the blood and triggers inflammatory responses, leading to hemolysis and vaso-occlusive crisis sequelae. Unfractionated heparin (UFH) and low-molecular weight heparins (LMWH) have been suggested as treatments to relieve coagulation complications in SCD. However, they are associated with bleeding complications after repeated dosing. An alternative sulfated nonanticoagulant heparin derivative (S-NACH) was previously reported to have none to low systemic anticoagulant activity and no bleeding side effects, and it interfered with P-selectindependent binding of sickle cells to endothelial cells, with concomitant decrease in the levels of adhesion biomarkers in SCD mice. S-NACH has been further engineered and structurally enhanced to bind with and modify HbS to directly inhibit sickling, thus employing a multimodal approach. Here, we show that S-NACH can (i) directly engage in Schiff-base reactions with HbS to decrease RBC sickling under both normoxia and hypoxia in vitro, ii) prolong the survival of SCD mice under hypoxia, and (iii) regulate the altered steady state levels of pro- and antiinflammatory cytokines. Thus, our proof of concept in vitro and in vivo preclinical studies demonstrate that the multimodal S-NACH is a highly promising candidate for development into an improved and optimized alternative to LMWHs for the treatment of patients with SCD.

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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

10.26434/chemrxiv.13634021.v1 ◽

2021 ◽

Author(s):

Klara Markova ◽

Antonin Kunka ◽

Klaudia Chmelova ◽

Martin Havlasek ◽

Petra Babkova ◽

...

Keyword(s):

Protein Folding ◽

Protein Design ◽

Energy Landscape ◽

Single Point ◽

Three Dimensional ◽

Protein Stabilization ◽

Dimensional Structure ◽

Evolutionary Analysis ◽

Folding Energy

The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (Tm = 73.5°C; ΔTm > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.

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Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

10.26434/chemrxiv.13634021 ◽

2021 ◽

Author(s):

Klara Markova ◽

Antonin Kunka ◽

Klaudia Chmelova ◽

Martin Havlasek ◽

Petra Babkova ◽

...

Keyword(s):

Protein Folding ◽

Protein Design ◽

Energy Landscape ◽

Single Point ◽

Three Dimensional ◽

Protein Stabilization ◽

Dimensional Structure ◽

Evolutionary Analysis ◽

Folding Energy

The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (Tm = 73.5°C; ΔTm > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively in vivo. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.

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