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 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.

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 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 A De novo Peptide from a High Throughput Peptide Library Blocks Myosin A -MTIP Complex Formation in Plasmodium falciparum

2020 ◽  
Vol 21 (17) ◽  
pp. 6158
Author(s):  
Zill e Anam ◽  
Nishant Joshi ◽  
Sakshi Gupta ◽  
Preeti Yadav ◽  
Ayushi Chaurasiya ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
De Novo ◽  
Peptide Library ◽  
Tail Domain ◽  
Interacting Protein ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
De Novo Peptide

Apicomplexan parasites, through their motor machinery, produce the required propulsive force critical for host cell-entry. The conserved components of this so-called glideosome machinery are myosin A and myosin A Tail Interacting Protein (MTIP). MTIP tethers myosin A to the inner membrane complex of the parasite through 20 amino acid-long C-terminal end of myosin A that makes direct contacts with MTIP, allowing the invasion of Plasmodium falciparum in erythrocytes. Here, we discovered through screening a peptide library, a de-novo peptide ZA1 that binds the myosin A tail domain. We demonstrated that ZA1 bound strongly to myosin A tail and was able to disrupt the native myosin A tail MTIP complex both in vitro and in vivo. We then showed that a shortened peptide derived from ZA1, named ZA1S, was able to bind myosin A and block parasite invasion. Overall, our study identified a novel anti-malarial peptide that could be used in combination with other antimalarials for blocking the invasion of Plasmodium falciparum.

The capactins, a class of proteins that cap the ends of actin filaments

1982 ◽  
Vol 299 (1095) ◽  
pp. 263-273 ◽  
Keyword(s):  
Skeletal Muscle ◽  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Preliminary Evidence ◽  
The Other ◽  
Cellular Regulation ◽  
Filament Assembly ◽  
Filament Bundles

A number of proteins that bind specifically to the barbed ends of actin filaments in a cytochalasin-like manner have been purified to various degrees from a variety of muscle and non-muscle cells and tissues. Preliminary evidence also indicates that proteins that interact with the pointed ends of filaments are present in skeletal muscle. Because of their ability to cap one or the other end of an actin filament, we have designated this class of proteins as the ‘capactins’. On the basis of their effect on actin filament assembly and interaction in vitro , we propose that the capactins play important roles in cellular regulation of actin-based cytoskeletal and contractile functions. Our finding that the disappearance of actin filament bundles in virally transformed fibroblasts can be correlated with an increase in capactin activity in the extracts of these cells is consistent with this hypothesis.

scholarly journals The rod domain of NF-L determines neurofilament architecture, whereas the end domains specify filament assembly and network formation.

1993 ◽  
Vol 123 (6) ◽  
pp. 1517-1533 ◽  
Author(s):  
S Heins ◽  
P C Wong ◽  
S Müller ◽  
K Goldie ◽  
D W Cleveland ◽  
...  
Keyword(s):  
Network Formation ◽  
Molecular Model ◽  
Tail Domain ◽  
Lateral Association ◽  
Filament Assembly ◽  
Head Domain ◽  
Scanning Transmission ◽  
Bona Fide ◽  
10 Nm Filaments

Neurofilaments, assembled from NF-L, NF-M, and NF-H subunits, are the most abundant structural elements in myelinated axons. Although all three subunits contain a central, alpha-helical rod domain thought to mediate filament assembly, only NF-L self-assembles into 10-nm filaments in vitro. To explore the roles of the central rod, the NH2-terminal head and the COOH-terminal tail domain in filament assembly, full-length, headless, tailless, and rod only fragments of mouse NF-L were expressed in bacteria, purified, and their structure and assembly properties examined by conventional and scanning transmission electron microscopy (TEM and STEM). These experiments revealed that in vitro assembly of NF-L into bona fide 10-nm filaments requires both end domains: whereas the NH2-terminal head domain promotes lateral association of protofilaments into protofibrils and ultimately 10-nm filaments, the COOH-terminal tail domain controls lateral assembly of protofilaments so that it terminates at the 10-nm filament level. Hence, the two end domains of NF-L have antagonistic effects on the lateral association of protofilaments into higher-order structures, with the effect of the COOH-terminal tail domain being dominant over that of the NH2-terminal head domain. Consideration of the 21-nm axial beading commonly observed with 10-nm filaments, the approximate 21-nm axial periodicity measured on paracrystals, and recent cross-linking data combine to support a molecular model for intermediate filament architecture in which the 44-46-nm long dimer rods overlap by 1-3-nm head-to-tail, whereas laterally they align antiparallel both unstaggered and approximately half-staggered.

scholarly journals Filensin and phakinin form a novel type of beaded intermediate filaments and coassemble de novo in cultured cells.

1996 ◽  
Vol 132 (4) ◽  
pp. 643-655 ◽  
Author(s):  
G Goulielmos ◽  
F Gounari ◽  
S Remington ◽  
S Müller ◽  
M Häner ◽  
...  
Keyword(s):  
Cultured Cells ◽  
De Novo ◽  
Spatial Arrangement ◽  
Scanning Transmission ◽  
Central Filament ◽  
Filament Bundles ◽  
Specific Proteins ◽  
Cytoskeletal Networks

The fiber cells of the eye lens possess a unique cytoskeletal system known as the "beaded-chain filaments" (BFs). BFs consist of filensin and phakinin, two recently characterized intermediate filament (IF) proteins. To examine the organization and the assembly of these heteropolymeric IFs, we have performed a series of in vitro polymerization studies and transfection experiments. Filaments assembled from purified filensin and phakinin exhibit the characteristic 19-21-nm periodicity seen in many types of IFs upon low angle rotary shadowing. However, quantitative mass-per-length (MPL) measurements indicate that filensin/phakinin filaments comprise two distinct and dissociable components: a core filament and a peripheral filament moiety. Consistent with a nonuniform organization, visualization of unfixed and unstained specimens by scanning transmission electron microscopy (STEM) reveals the the existence of a central filament which is decorated by regularly spaced 12-15-nm-diam beads. Our data suggest that the filamentous core is composed of phakinin, which exhibits a tendency to self-assemble into filament bundles, whereas the beads contain filensin/phakinin hetero-oligomers. Filensin and phakinin copolymerize and form filamentous structures when expressed transiently in cultured cells. Experiments in IF-free SW13 cells reveal that coassembly of the lens-specific proteins in vivo does not require a preexisting IF system. In epithelial MCF-7 cells de novo forming filaments appear to grow from distinct foci and organize as thick, fibrous laminae which line the plasma membrane and the nuclear envelope. However, filament assembly in CHO and SV40-transformed lens-epithelial cells (both of which are fibroblast-like) yields radial networks which codistribute with the endogenous vimentin IFs. These observations document that the filaments formed by lens-specific IF proteins are structurally distinct from ordinary cytoplasmic IFs. Furthermore, the results suggest that the spatial arrangement of filensin/phakinin filaments in vivo is subject to regulation by host-specific factors. These factors may involve cytoskeletal networks (e.g., vimentin IFs) and/or specific sites associated with the cellular membranes.

scholarly journals Prolonged Idasanutlin (RG7388) Treatment Leads to the Generation of p53-Mutated Cells

Cancers ◽  
2018 ◽  
Vol 10 (11) ◽  
pp. 396 ◽  
Author(s):  
Lukasz Skalniak ◽  
Justyna Kocik ◽  
Justyna Polak ◽  
Anna Skalniak ◽  
Monika Rak ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Clinical Trials ◽  
Cell Cycle Arrest ◽  
De Novo ◽  
Phase Iii ◽  
Prolonged Treatment ◽  
Secondary Resistance ◽  
Cycle Arrest ◽  
Mcf 7

The protein p53 protects the organism against carcinogenic events by the induction of cell cycle arrest and DNA repair program upon DNA damage. Virtually all cancers inactivate p53 either by mutations/deletions of the TP53 gene or by boosting negative regulation of p53 activity. The overexpression of MDM2 protein is one of the most common mechanisms utilized by p53wt cancers to keep p53 inactive. Inhibition of MDM2 action by its antagonists has proved its anticancer potential in vitro and is now tested in clinical trials. However, the prolonged treatment of p53wt cells with MDM2 antagonists leads to the development of secondary resistance, as shown first for Nutlin-3a, and later for three other small molecules. In the present study, we show that secondary resistance occurs also after treatment of p53wt cells with idasanutlin (RG7388, RO5503781), which is the only MDM2 antagonist that has passed phase II and entered phase III clinical trials, so far. Idasanutlin strongly activates p53, as evidenced by the induction of p21 expression and potent cell cycle arrest in all the three cell lines tested, i.e., MCF-7, U-2 OS, and SJSA-1. Notably, apoptosis was induced only in SJSA-1 cells, while MCF-7 and U-2 OS cells were able to restore the proliferation upon the removal of idasanutlin. Moreover, idasanutlin-treated U-2 OS cells could be cultured for long time periods in the presence of the drug. This prolonged treatment led to the generation of p53-mutated resistant cell populations. This resistance was generated de novo, as evidenced by the utilization of monoclonal U-2 OS subpopulations. Thus, although idasanutlin presents much improved activities compared to its precursor, it displays the similar weaknesses, which are limited elimination of cancer cells and the generation of p53-mutated drug-resistant subpopulations.

scholarly journals Reconstitution of cytokeratin filaments in vitro: further evidence for the role of nonhelical peptides in filament assembly.

1984 ◽  
Vol 99 (5) ◽  
pp. 1590-1597 ◽  
Author(s):  
J J Sauk ◽  
M Krumweide ◽  
D Cocking-Johnson ◽  
J G White
Keyword(s):  
Self Assembly ◽  
Structural Information ◽  
Alpha Helix ◽  
Structural Domains ◽  
Filament Assembly ◽  
Low Salt ◽  
Turbidity Increase ◽  
Cytokeratin Filaments

The in vitro renaturation and assembly of cytokeratin molecules to form intermediate filaments (IF) illustrates that these molecules contain all of the structural information necessary for IF information. These molecules contain nine structural domains: the amino- and carboxyterminal extra helical regions, and three conserved extra helical segments that separate four helical rod-like domains. Chymotrypsin treatment of these molecules removes the end-peptide domains and inhibits the self-assembly process. We have examined the renaturation and assembly of cytokeratin molecules using solution conditions that favor the presence of intermediate forms of IF organization. Dialysis against low salt buffers revealed the presence of bead-like chains of filaments in which the 6-8-nm beads are separated by a distance of 21 nm. These data suggest that a lateral stagger of protofilaments was among the primary events in IF assembly. Chymotrypsin-modified cytokeratin enriched for alpha-helix barely initiated a turbidity increase at conditions favoring self-assembly. Addition of small amounts of intact cytokeratin accelerated the rate and extent of this reaction. These results indicate that the nonhelical peptides on intact cytokeratin potentiate the assembly of IF by orientating the stagger of laterally associated protofilaments.

Einfluß der Glucose auf die [14C] Thymidin-Einbaurate von Ehrlich-Ascitescarcinomzellen in vitro

1969 ◽  
Vol 08 (02) ◽  
pp. 196-206 ◽  
Author(s):  
Dieter. Kummer
Keyword(s):  
De Novo

ZusammenfassungIn nahezu glucosefreier Suspension von Ehrlich-Ascitescarcinomzellen bewirkt die Zufuhr von Glucose 2,5 × 10–4 bis 10–2 M:1. Hemmung der [14C] Thymidin-Einbaurate in die Zellen.2. Aktivierung des Ribonucleotid-Reductase-Systems und damit Stimulierung der Desoxyribonucleotidsynthese (auch der Thymidintriphosphat-de-novo-Synthese).3. Blockierung der Thymidinkinase über Endprodukthemmung, wodurch die Minderung des [14C] Thymidin-Einbaus in die Zellen erklärbar ist.

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