scholarly journals Cryo-EM structures of CTP synthase filaments reveal mechanism of pH-sensitive assembly during budding yeast starvation

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Jesse M Hansen ◽  
Avital Horowitz ◽  
Eric M Lynch ◽  
Daniel P Farrell ◽  
Joel Quispe ◽  
...  
Keyword(s):  
Metabolic Enzymes ◽  
Ph Sensitive ◽  
Starvation Response ◽  
Filament Assembly ◽  
Assembly Mechanism ◽  
Ctp Synthase ◽  
Filament Bundles ◽  
Cytoplasmic Acidification

Many metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or pH-insensitive mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

scholarly journals Cryo-EM Structures of CTP Synthase Filaments Reveal Mechanism of pH-Sensitive Assembly During Budding Yeast Starvation

2021 ◽  
Author(s):  
Jesse M Hansen ◽  
Avital Horowitz ◽  
Eric M Lynch ◽  
Daniel P Farrell ◽  
Joel Quispe ◽  
...  
Keyword(s):  
Metabolic Enzymes ◽  
Ph Sensitive ◽  
Starvation Response ◽  
Filament Assembly ◽  
Assembly Mechanism ◽  
Ctp Synthase ◽  
Filament Bundles ◽  
Cytoplasmic Acidification

ABSTRACTMany metabolic enzymes self-assemble into micron-scale filaments to organize and regulate metabolism. The appearance of these assemblies often coincides with large metabolic changes as in development, cancer, and stress. Yeast undergo cytoplasmic acidification upon starvation, triggering the assembly of many metabolic enzymes into filaments. However, it is unclear how these filaments assemble at the molecular level and what their role is in the yeast starvation response. CTP Synthase (CTPS) assembles into metabolic filaments across many species. Here, we characterize in vitro polymerization and investigate in vivo consequences of CTPS assembly in yeast. Cryo-EM structures reveal a pH-sensitive assembly mechanism and highly ordered filament bundles that stabilize an inactive state of the enzyme, features unique to yeast CTPS. Disruption of filaments in cells with non-assembly or hyper-assembly mutations decreases growth rate, reflecting the importance of regulated CTPS filament assembly in homeotstasis.

scholarly journals A potential role for the COOH-terminal domain in the lateral packing of type III intermediate filaments.

1991 ◽  
Vol 114 (4) ◽  
pp. 773-786 ◽  
Author(s):  
P D Kouklis ◽  
T Papamarcaki ◽  
A Merdes ◽  
S D Georgatos
Keyword(s):  
Potential Role ◽  
Type Iii ◽  
Filament Assembly ◽  
Filament Bundles ◽  
Self Association ◽  
Specific Association ◽  
A Site ◽  
Idiotypic Antibody

To identify sites of self-association in type III intermediate filament (IF) proteins, we have taken an "anti-idiotypic antibody" approach. A mAb (anti-Ct), recognizing a similar feature near the end of the rod domain of vimentin, desmin, and peripherin (epsilon site or epsilon epitope), was characterized. Anti-idiotypic antibodies, generated by immunizing rabbits with purified anti-Ct, recognize a site (presumably "complementary" to the epsilon epitope) common among vimentin, desmin, and peripherin (beta site or beta epitope). The beta epitope is represented in a synthetic peptide (PII) modeled after the 30 COOH-terminal residues of peripherin, as seen by comparative immunoblotting assays. Consistent with the idea of an association between the epsilon and the beta site, PII binds in vitro to intact IF proteins and fragments containing the epsilon epitope, but not to IF proteins that do not react with anti-Ct. Microinjection experiments conducted in vivo and filament reconstitution assays carried out in vitro further demonstrate that "uncoupling" of this site-specific association (by competition with PII or anti-Ct) interferes with normal IF architecture, resulting in the formation of filaments and filament bundles with diameters much greater than that of the normal IFs. These thick fibers are very similar to the ones observed previously when a derivative of desmin missing 27 COOH-terminal residues was assembled in vitro (Kaufmann, E., K. Weber, and N. Geisler. 1985. J. Mol. Biol. 185:733-742). As a molecular explanation, we propose here that the epsilon and the beta sites of type III IF proteins are "complementary" and associate during filament assembly. As a result of this association, we further postulate the formation of a surface-exposed "loop" or "hairpin" structure that may sterically prevent inappropriate filament-filament aggregation and regulate filament thickness.

RGD(Arg-Gly-Asp) internalized docetaxel-loaded pH sensitive liposomes: Preparation, characterization and antitumor efficacy in vivo and in vitro

2016 ◽  
Vol 147 ◽  
pp. 90-99 ◽  
Author(s):  
Tiantian Zuo ◽  
Yuanyuan Guan ◽  
Minglu Chang ◽  
Fang Zhang ◽  
Shanshan Lu ◽  
...  
Keyword(s):  
Ph Sensitive ◽  
Antitumor Efficacy

scholarly journals Effects of cytoplasmic acidification on clathrin lattice morphology.

1989 ◽  
Vol 108 (2) ◽  
pp. 401-411 ◽  
Author(s):  
J Heuser
Keyword(s):  
Plasma Membrane ◽  
Cultured Cells ◽  
Membrane Receptors ◽  
The Other ◽  
Culture Dish ◽  
Initial Curvature ◽  
Internal Ph ◽  
Cytoplasmic Acidification

Reducing the internal pH of cultured cells by several different protocols that block endocytosis is found to alter the structure of clathrin lattices on the inside of the plasma membrane. Lattices curve inward until they become almost spherical yet remain stubbornly attached to the membrane. Also, the lattices bloom empty "microcages" of clathrin around their edges. Correspondingly, broken-open cells bathed in acidified media demonstrate similar changes in clathrin lattices. Acidification accentuates the normal tendency of lattices to round up in vitro and also stimulates them to nucleate microcage formation from pure solutions of clathrin. On the other hand, several conditions that also inhibit endocytosis have been found to create, instead of unusually curved clathrin lattices with extraneous microcages, a preponderance of unusually flat lattices. These treatments include pH-"clamping" cells at neutrality with nigericin, swelling cells with hypotonic media, and sticking cells to the surface of a culture dish with soluble polylysine. Again, the unusually flat lattices in such cells display a tendency to round up and to nucleate clathrin microcage formation during subsequent in vitro acidification. This indicates that regardless of the initial curvature of clathrin lattices, they all display an ability to grow and increase their curvature in vitro, and this is enhanced by lowering ambient pH. Possibly, clathrin lattice growth and curvature in vivo may also be stimulated by a local drop in pH around clusters of membrane receptors.

Contributions of the structural domains of filensin in polymer formation and filament distribution

1996 ◽  
Vol 109 (2) ◽  
pp. 447-456
Author(s):  
G. Goulielmos ◽  
S. Remington ◽  
F. Schwesinger ◽  
S.D. Georgatos ◽  
F. Gounari
Keyword(s):  
De Novo ◽  
Chimeric Proteins ◽  
Tail Domain ◽  
Structural Domains ◽  
Filament Assembly ◽  
Polymer Formation ◽  
Filament Bundles ◽  
Specific Contribution ◽  
Mcf 7

Filensin and phakinin constitute the subunits of a heteropolymeric, lens-specific intermediate filament (IF) system known as the beaded-chain filaments (BFs). Since the rod of filensin is four heptads shorter than the rods of all other IF proteins, we decided to examine the specific contribution of this protein in filament assembly. For these purposes, we constructed chimeric proteins in which regions of filensin were exchanged with the equivalent ones of vimentin, a self-polymerizing IF protein. Our in vitro studies show that the filensin rod domain does not allow homopolymeric filament elongation. However, the filensin rod is necessary for co-polymerization of filensin with phakinin and seems to counteract the inherent tendency of the latter protein to homopolymerize into large, laterally associated filament bundles. Apart from the rod domain, the presence of an authentic or substituted tail domain in filensin is also essential for co-assembly with the naturally tail-less phakinin and formation of extended filaments in vitro. Finally, transfection experiments in CHO and MCF-7 cells show that the rod domain of filensin plays an important role in de novo filament formation and distribution. The same type of analysis further suggests that the end-domains of filensin interact with cell-specific, assembly-modulating factors.

scholarly journals pH-Sensitive Hydrazone-Linked Doxorubicin Nanogels via Polymeric-Activated Ester Scaffolds: Synthesis, Assembly, and In Vitro and In Vivo Evaluation in Tumor-Bearing Zebrafish

2018 ◽  
Vol 30 (23) ◽  
pp. 8587-8596 ◽  
Author(s):  
Alexandra Van Driessche ◽  
Agnese Kocere ◽  
Hannelien Everaert ◽  
Lutz Nuhn ◽  
Simon Van Herck ◽  
...  
Keyword(s):  
Ph Sensitive ◽  
In Vivo Evaluation ◽  
Tumor Bearing

scholarly journals Proteomic and 3D structure analyses highlight the C/D box snoRNP assembly mechanism and its control

2014 ◽  
Vol 207 (4) ◽  
pp. 463-480 ◽  
Author(s):  
Jonathan Bizarro ◽  
Christophe Charron ◽  
Séverine Boulon ◽  
Belinda Westman ◽  
Bérengère Pradet-Balade ◽  
...  
Keyword(s):  
Isotope Labeling ◽  
Core Protein ◽  
3D Structure ◽  
Assembly Mechanism ◽  
Core Proteins ◽  
Assembly Factors ◽  
Assembly Factor

In vitro, assembly of box C/D small nucleolar ribonucleoproteins (snoRNPs) involves the sequential recruitment of core proteins to snoRNAs. In vivo, however, assembly factors are required (NUFIP, BCD1, and the HSP90–R2TP complex), and it is unknown whether a similar sequential scheme applies. In this paper, we describe systematic quantitative stable isotope labeling by amino acids in cell culture proteomic experiments and the crystal structure of the core protein Snu13p/15.5K bound to a fragment of the assembly factor Rsa1p/NUFIP. This revealed several unexpected features: (a) the existence of a protein-only pre-snoRNP complex containing five assembly factors and two core proteins, 15.5K and Nop58; (b) the characterization of ZNHIT3, which is present in the protein-only complex but gets released upon binding to C/D snoRNAs; (c) the dynamics of the R2TP complex, which appears to load/unload RuvBL AAA+ adenosine triphosphatase from pre-snoRNPs; and (d) a potential mechanism for preventing premature activation of snoRNP catalytic activity. These data provide a framework for understanding the assembly of box C/D snoRNPs.

Single-unit pH-sensitive double-barreled microelectrodes for extracellular use

1984 ◽  
Vol 57 (3) ◽  
pp. 907-912
Author(s):  
S. Javaheri ◽  
A. De Hemptinne ◽  
I. Leusen
Keyword(s):  
Direct Current ◽  
Single Unit ◽  
Extracellular Fluid ◽  
Ph Sensitive ◽  
Current Potential

The purpose of this study is to systematically describe the construction of pH-sensitive double-barreled microelectrodes for extracellular use. The most important advantages of these microelectrodes are as follows: the reference and the pH barrels are next to each other, and therefore the measured pH is not affected by asymmetric or slowly spreading direct current potential. The diameter of the tip of the microelectrodes is between 7 and 35 micron. These pH-sensitive microelectrodes are generally stable and Nernstian. They can be used repeatedly both in vivo and in vitro to measure tissue extracellular fluid pH. Some applications are described.

scholarly journals pH-Sensitive Ratiometric Fluorescent Probe for Evaluation of Tumor Treatments

Materials ◽  
2019 ◽  
Vol 12 (10) ◽  
pp. 1632
Author(s):  
Peisen Zhang ◽  
Junli Meng ◽  
Yingying Li ◽  
Zihua Wang ◽  
Yi Hou
Keyword(s):  
Ph Sensitive ◽  
Fluorescent Imaging ◽  
Tat Peptide ◽  
Cancer Drugs ◽  
Ph Response ◽  
In Vivo Experiments ◽  
Anti Cancer ◽  
Amidation Reaction

Determining therapeutic efficacy is critical for tumor precision theranostics. In order to monitor the efficacy of anti-cancer drugs (e.g., Paclitaxel), a pH-sensitive ratiometric fluorescent imaging probe was constructed. The pH-sensitive ratiometric fluorescent dye ANNA was covalently coupled to the N-terminal of the cell-penetrating TAT peptide through an amidation reaction (TAT-ANNA). The in vitro cellular experiments determined that the TAT-ANNA probe could penetrate the cell membrane and image the intracellular pH in real time. The in vivo experiments were then carried out, and the ratiometric pH response to the state of the tumor was recorded immediately after medication. The TAT-ANNA probe was successfully used to monitor the pharmacodynamics of anti-cancer drugs in vivo.

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