scholarly journals Mutations of Oncoprotein 18/Stathmin Identify Tubulin-Directed Regulatory Activities Distinct from Tubulin Association

1999 ◽  
Vol 19 (3) ◽  
pp. 2242-2250 ◽  
Author(s):  
Niklas Larsson ◽  
Bo Segerman ◽  
Helena Melander Gradin ◽  
Ewa Wandzioch ◽  
Lynne Cassimeris ◽  
...  
Keyword(s):  
Complex Formation ◽  
Dynamic Properties ◽  
Coiled Coil ◽  
Leukemia Cells ◽  
Tubulin Polymerization ◽  
Phosphorylation Sites ◽  
Wild Type ◽  
Coiled Coil Region ◽  
Oncoprotein 18 ◽  
Heptad Repeats

ABSTRACT Oncoprotein 18/stathmin (Op18) is a recently identified phosphorylation-responsive regulator of the microtubule (MT) system. It was originally proposed that Op18 specifically regulates dynamic properties of MTs by associating with tubulin, but it has subsequently been proposed that Op18 acts simply by sequestering of tubulin heterodimers. We have dissected the mechanistic action of Op18 by generation of two distinct classes of mutants. One class has interruptions of the heptad repeats of a potential coiled-coil region of Op18, and the other involves substitution at all four phosphorylation sites with negatively charged Glu residues. Both types of mutation result in Op18 proteins with a limited decrease in tubulin complex formation. However, the MT-destabilizing activities of the coiled-coil mutants are more severely reduced in transfected leukemia cells than those of the Glu-substituted Op18 derivative, providing evidence for tubulin-directed regulatory activities distinct from tubulin complex formation. Analysis of Op18-mediated regulation of tubulin GTPase activity and taxol-promoted tubulin polymerization showed that while wild-type and Glu-substituted Op18 derivatives are active, the coiled-coil mutants are essentially inactive. This suggests that Op18-tubulin contact involves structural motifs that deliver a signal of regulatory importance to the MT system.

scholarly journals 2066 Functional characterization of mutant BRCA1

2018 ◽  
Vol 2 (S1) ◽  
pp. 13-13
Author(s):  
John Barrows ◽  
David Long
Keyword(s):  
Functional Characterization ◽  
Coiled Coil ◽  
Wild Type ◽  
Coiled Coil Region ◽  
Egg Extracts ◽  
Study Population ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

scholarly journals Mechanistic insights into the interactions of dynein regulator Ndel1 with neuronal ankyrins and implications in polarity maintenance

2019 ◽  
Vol 117 (2) ◽  
pp. 1207-1215 ◽  
Author(s):  
Jin Ye ◽  
Jianchao Li ◽  
Fei Ye ◽  
Yan Zhang ◽  
Mingjie Zhang ◽  
...  
Keyword(s):  
Complex Formation ◽  
Hydrophobic Interactions ◽  
Coiled Coil ◽  
Structural Data ◽  
Giant Neuron ◽  
Ankyrin G ◽  
Regulator Protein ◽  
Coiled Coil Region ◽  
Mechanistic Basis ◽  
Cargo Sorting

Ankyrin-G (AnkG), a highly enriched scaffold protein in the axon initial segment (AIS) of neurons, functions to maintain axonal polarity and the integrity of the AIS. At the AIS, AnkG regulates selective intracellular cargo trafficking between soma and axons via interaction with the dynein regulator protein Ndel1, but the molecular mechanism underlying this binding remains elusive. Here we report that Ndel1’s C-terminal coiled-coil region (CT-CC) binds to giant neuron-specific insertion regions present in both AnkG and AnkB with 2:1 stoichiometry. The high-resolution crystal structure of AnkB in complex with Ndel1 CT-CC revealed the detailed molecular basis governing the AnkB/Ndel1 complex formation. Mechanistically, AnkB binds with Ndel1 by forming a stable 5-helix bundle dominated by hydrophobic interactions spread across 6 distinct interaction layers. Moreover, we found that AnkG is essential for Ndel1 accumulation at the AIS. Finally, we found that cargo sorting at the AIS can be disrupted by blocking the AnkG/Ndel1 complex formation using a peptide designed based on our structural data. Collectively, the atomic structure of the AnkB/Ndel1 complex together with studies of cargo sorting through the AIS establish the mechanistic basis for AnkG/Ndel1 complex formation and for the maintenance of axonal polarity. Our study will also be valuable for future studies of the interaction between AnkB and Ndel1 perhaps at distal axonal cargo transport.

scholarly journals Human Immunodeficiency Virus (HIV) gp41 Escape Mutants: Cross-Resistance to Peptide Inhibitors of HIV Fusion and Altered Receptor Activation of gp120

2005 ◽  
Vol 79 (8) ◽  
pp. 4774-4781 ◽  
Author(s):  
Emmanuel Desmezieres ◽  
Nidhi Gupta ◽  
Russell Vassell ◽  
Yong He ◽  
Keith Peden ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Coiled Coil ◽  
Receptor Activation ◽  
Peptide Inhibitors ◽  
Dominant Negative ◽  
Heptad Repeat ◽  
Cross Resistance ◽  
Wild Type ◽  
Immunodeficiency Virus ◽  
Heptad Repeats

ABSTRACT Human immunodeficiency virus (HIV) infects cells by fusing with cellular membranes. Fusion occurs when the envelope glycoprotein (Env) undergoes conformational changes while binding to cellular receptors. Fusogenic changes involve assembly of two heptad repeats in the ectodomain of the gp41 transmembrane subunit to form a six-helix bundle (6HB), consisting of a trimeric N heptad repeat (N-HR) coiled-coil core with three antiparallel C heptad repeats (C-HRs) that pack in the coiled-coil grooves. Peptides corresponding to the N-and C-HRs (N and C peptides, respectively) interfere with formation of the 6HB in a dominant-negative manner and are emerging as a new class of antiretroviral therapeutics for treating HIV infection. We generated an escape mutant virus with resistance to an N peptide and show that early resistance involved two mutations, one each in the N- and C-HRs. The mutations conferred resistance not only to the selecting N peptide but also to C peptides, as well as other types of N-peptide inhibitors. Moreover, the N-HR mutation altered sensitivity to soluble CD4. Biophysical studies suggest that the 6HB with the resistance mutations is more stable than the wild-type 6HB and the 6HB formed by inhibitor binding to either wild-type or mutant C-HR. These findings provide new insights into potential mechanisms of resistance to HIV peptide fusion inhibitors and dominant-negative inhibitors in general. The results are discussed in the context of current models of Env-mediated membrane fusion.

scholarly journals Relevance of Ebola virus VP35 homo-dimerization on the type I interferon cascade inhibition

2019 ◽  
Vol 27 ◽  
pp. 204020661988922 ◽  
Author(s):  
Francesco Di Palma ◽  
Gian Luca Daino ◽  
Venkata Krishnan Ramaswamy ◽  
Angela Corona ◽  
Aldo Frau ◽  
...  
Keyword(s):  
Ebola Virus ◽  
Type I Interferon ◽  
Coiled Coil ◽  
Coarse Grained ◽  
Type I ◽  
Wild Type ◽  
Terminal Domain ◽  
Coiled Coil Region ◽  
Interferon Antagonism

Ebola virus high lethality relies on its ability to efficiently bypass the host innate antiviral response, which senses the viral dsRNA through the RIG-I receptor and induces type I interferon α/β production. In the bypassing action, the Ebola virus protein VP35 plays a pivotal role at multiple levels of the RIG-I cascade, masking the viral 5′-triphosphorylated dsRNA from RIG-I, and interacting with other cascade components. The VP35 type I interferon inhibition is exerted by the C-terminal domain, while the N-terminal domain, containing a coiled-coil region, is primarily required for oligomerization. However, mutations at key VP35 residues L90/93/107A (VP35-3m) in the coiled-coil region were reported to affect oligomerization and reduce type I interferon antagonism, indicating a possible but unclear role of homo-oligomerization on VP35 interaction with the RIG-I pathway components. In this work, we investigated the VP35 dimerization thermodynamics and its contribution to type I interferon antagonism by computational and biological methods. Focusing on the coiled-coil region, we combined coarse-grained and all-atom simulations on wild type VP35 and VP35-3m homo-dimerization. According to our results, wild type VP35 coiled-coil is able to self-assemble into dimers, while VP35-3m coiled-coil shows poor propensity to even dimerize. Free-energy calculations confirmed the key role of L90, L93 and L107 in stabilizing the coiled-coil homo-dimeric structure. In vitro type I interferon antagonism studies, using full-length wild type VP35 and VP35-3m, revealed that VP35 homo-dimerization is an essential preliminary step for dsRNA binding, which appears to be the main factor of the VP35 RIG-I cascade inhibition, while it is not essential to block the other steps.

scholarly journals N- and C-terminal Gln3–Tor1 interaction sites: one acting negatively and the other positively to regulate nuclear Gln3 localization

Genetics ◽  
2021 ◽  
Vol 217 (4) ◽  
Author(s):  
Jennifer J Tate ◽  
Rajendra Rai ◽  
Claudio De Virgilio ◽  
Terrance G Cooper
Keyword(s):  
Coiled Coil ◽  
Nitrogen Sources ◽  
Growth Conditions ◽  
Nitrogen Catabolite Repression ◽  
Wild Type ◽  
Interaction Site ◽  
Interaction Sites ◽  
Coiled Coil Region ◽  
Transcription Factor Regulation ◽  
Α Helix

Abstract Gln3 activates Nitrogen Catabolite Repression, NCR-sensitive expression of the genes required for Saccharomyces cerevisiae to scavenge poor nitrogen sources from its environment. The global TorC1 kinase complex negatively regulates nuclear Gln3 localization, interacting with an α-helix in the C-terminal region of Gln3, Gln3656–666. In nitrogen replete conditions, Gln3 is sequestered in the cytoplasm, whereas when TorC1 is down-regulated, in nitrogen restrictive conditions, Gln3 migrates into the nucleus. In this work, we show that the C-terminal Gln3–Tor1 interaction site is required for wild type, rapamycin-elicited, Sit4-dependent nuclear Gln3 localization, but not for its dephosphorylation. In fact, truncated Gln31-384 can enter the nucleus in the absence of Sit4 in both repressive and derepressive growth conditions. However, Gln31-384 can only enter the nucleus if a newly discovered second positively-acting Gln3–Tor1 interaction site remains intact. Importantly, the N- and C-terminal Gln3–Tor1 interaction sites function both autonomously and collaboratively. The N-terminal Gln3–Tor1 interaction site, previously designated Gln3URS contains a predicted α-helix situated within an unstructured coiled-coil region. Eight of the thirteen serine/threonine residues in the Gln3URS are dephosphorylated 3–15-fold with three of them by 10–15-fold. Substituting phosphomimetic aspartate for serine/threonine residues in the Gln3 URS abolishes the N-terminal Gln3–Tor1 interaction, rapamycin-elicited nuclear Gln3 localization, and ½ of the derepressed levels of nuclear Gln3 localization. Cytoplasmic Gln3 sequestration in repressive conditions, however, remains intact. These findings further deconvolve the mechanisms that achieve nitrogen-responsive transcription factor regulation downstream of TorC1.

The distribution of antigen on the surface of RBL-2H3 rat basophilic leukemia cells observed by back-scattered electron imaging

1986 ◽  
Vol 44 ◽  
pp. 274-275
Author(s):  
R.F. Stump ◽  
J.R. Pfeiffer ◽  
JC. Seagrave ◽  
D. Huskisson ◽  
J.M. Oliver
Keyword(s):  
Cell Surface ◽  
Dynamic Properties ◽  
Leukemia Cells ◽  
Antigen Binding ◽  
Scattered Electron ◽  
Ige Receptor ◽  
Receptor Complexes ◽  
Rat Basophilic Leukemia Cells ◽  
Electron Imaging ◽  
Basophilic Leukemia

In RBL-2H3 rat basophilic leukemia cells, antigen binding to cell surface IgE-receptor complexes stimulates the release of inflammatory mediators and initiates a series of membrane and cytoskeletal events including a transformation of the cell surface from a microvillous to a lamellar topography. It is likely that dynamic properties of the IgE receptor contribute to the activation of these responses. Fewtrell and Metzger have established that limited crosslinking of IgE-receptor complexes is essential to trigger secretion. In addition, Baird and colleagues have reported that antigen binding causes a rapid immobilization of IgE-receptor complexes, and we have demonstrated an apparent increase with time in the affinity of IgE-receptor complexes for antigen.

scholarly journals FGFR3 overexpression is a useful detection tool for FGFR3 fusions and sequence variations in glioma

2020 ◽  
Author(s):  
Jens Schittenhelm ◽  
Lukas Ziegler ◽  
Jan Sperveslage ◽  
Michel Mittelbronn ◽  
David Capper ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Growth Factor Receptor ◽  
Gene Mutations ◽  
Diagnostic Tools ◽  
Wild Type ◽  
Rt Pcr ◽  
Fgfr3 Gene ◽  
Detection Tool ◽  
Sequence Variations ◽  
Gene Panel Sequencing

Abstract Background Fibroblast growth factor receptor (FGFR) inhibitors are currently used in clinical development. A subset of glioblastomas carries gene fusion of FGFR3 and transforming acidic coiled-coil protein 3. The prevalence of other FGFR3 alterations in glioma is currently unclear. Methods We performed RT-PCR in 101 glioblastoma samples to detect FGFR3-TACC3 fusions (“RT-PCR cohort”) and correlated results with FGFR3 immunohistochemistry (IHC). Further, we applied FGFR3 IHC in 552 tissue microarray glioma samples (“TMA cohort”) and validated these results in two external cohorts with 319 patients. Gene panel sequencing was carried out in 88 samples (“NGS cohort”) to identify other possible FGFR3 alterations. Molecular modeling was performed on newly detected mutations. Results In the “RT-PCR cohort,” we identified FGFR3-TACC3 fusions in 2/101 glioblastomas. Positive IHC staining was observed in 73/1024 tumor samples of which 10 were strongly positive. In the “NGS cohort,” we identified FGFR3 fusions in 9/88 cases, FGFR3 amplification in 2/88 cases, and FGFR3 gene mutations in 7/88 cases in targeted sequencing. All FGFR3 fusions and amplifications and a novel FGFR3 K649R missense mutation were associated with FGFR3 overexpression (sensitivity and specificity of 93% and 95%, respectively, at cutoff IHC score > 7). Modeling of these data indicated that Tyr647, a residue phosphorylated as a part of FGFR3 activation, is affected by the K649R mutation. Conclusions FGFR3 IHC is a useful screening tool for the detection of FGFR3 alterations and could be included in the workflow for isocitrate dehydrogenase (IDH) wild-type glioma diagnostics. Samples with positive FGFR3 staining could then be selected for NGS-based diagnostic tools.

scholarly journals A microfluidic device enabling drug resistance analysis of leukemia cells via coupled dielectrophoretic detection and impedimetric counting

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yağmur Demircan Yalçın ◽  
Taylan Berkin Töral ◽  
Sertan Sukas ◽  
Ender Yıldırım ◽  
Özge Zorlu ◽  
...  
Keyword(s):  
Drug Resistance ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Drug Resistant ◽  
Wild Type ◽  
Gene Expressions ◽  
Before And After ◽  
The Difference ◽  
Selection Of ◽  
Resistant Cells

AbstractWe report the development of a lab-on-a-chip system, that facilitates coupled dielectrophoretic detection (DEP-D) and impedimetric counting (IM-C), for investigating drug resistance in K562 and CCRF-CEM leukemia cells without (immuno) labeling. Two IM-C units were placed upstream and downstream of the DEP-D unit for enumeration, respectively, before and after the cells were treated in DEP-D unit, where the difference in cell count gave the total number of trapped cells based on their DEP characteristics. Conductivity of the running buffer was matched the conductivity of cytoplasm of wild type K562 and CCRF-CEM cells. Results showed that DEP responses of drug resistant and wild type K562 cells were statistically discriminative (at p = 0.05 level) at 200 mS/m buffer conductivity and at 8.6 MHz working frequency of DEP-D unit. For CCRF-CEM cells, conductivity and frequency values were 160 mS/m and 6.2 MHz, respectively. Our approach enabled discrimination of resistant cells in a group by setting up a threshold provided by the conductivity of running buffer. Subsequent selection of drug resistant cells can be applied to investigate variations in gene expressions and occurrence of mutations related to drug resistance.

scholarly journals A Coiled-Coil Domain of Melanophilin Is Essential for Myosin Va Recruitment and Melanosome Transport in Melanocytes

2006 ◽  
Vol 17 (11) ◽  
pp. 4720-4735 ◽  
Author(s):  
Alistair N. Hume ◽  
Abul K. Tarafder ◽  
José S. Ramalho ◽  
Elena V. Sviderskaya ◽  
Miguel C. Seabra
Keyword(s):  
Coiled Coil ◽  
Binding Domain ◽  
Actin Binding ◽  
Binding Studies ◽  
Null Cell ◽  
Coiled Coil Region ◽  
Actin Binding Domain ◽  
Transport Assay ◽  
Myosin Va

Melanophilin (Mlph) regulates retention of melanosomes at the peripheral actin cytoskeleton of melanocytes, a process essential for normal mammalian pigmentation. Mlph is proposed to be a modular protein binding the melanosome-associated protein Rab27a, Myosin Va (MyoVa), actin, and microtubule end-binding protein (EB1), via distinct N-terminal Rab27a-binding domain (R27BD), medial MyoVa-binding domain (MBD), and C-terminal actin-binding domain (ABD), respectively. We developed a novel melanosome transport assay using a Mlph-null cell line to study formation of the active Rab27a:Mlph:MyoVa complex. Recruitment of MyoVa to melanosomes correlated with rescue of melanosome transport and required intact R27BD together with MBD exon F–binding region (EFBD) and unexpectedly a potential coiled-coil forming sequence within ABD. In vitro binding studies indicate that the coiled-coil region enhances binding of MyoVa by Mlph MBD. Other regions of Mlph reported to interact with MyoVa globular tail, actin, or EB1 are not essential for melanosome transport rescue. The strict correlation between melanosomal MyoVa recruitment and rescue of melanosome distribution suggests that stable interaction with Mlph and MyoVa activation are nondissociable events. Our results highlight the importance of the coiled-coil region together with R27BD and EFBD regions of Mlph in the formation of the active melanosomal Rab27a-Mlph-MyoVa complex.

scholarly journals The Alopecia Areata Phenotype Is Induced by the Water Avoidance Stress Test In cchcr1-Deficient Mice

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 840
Author(s):  
Qiaofeng Zhao ◽  
Satoshi Koyama ◽  
Nagisa Yoshihara ◽  
Atsushi Takagi ◽  
Etsuko Komiyama ◽  
...  
Keyword(s):  
Alopecia Areata ◽  
Gene Knockout ◽  
Stress Test ◽  
Coiled Coil ◽  
Risk Haplotype ◽  
Gene Expression Microarray ◽  
Wild Type ◽  
Gene Knockout Mice ◽  
Deficient Mice ◽  
Gene Expression Microarray Analysis

We recently discovered a nonsynonymous variant in the coiled-coil alpha-helical rod protein 1 (CCHCR1) gene within the alopecia areata (AA) risk haplotype. We also reported that the engineered mice with this risk allele exhibited. To investigate more about the involvement of the CCHCR1 gene in AA pathogenesis, we developed an AA model using C57BL/6N cchcr1 gene knockout mice. In this study, mice (6–8 weeks) were divided into two groups: cchcr1−/− mice and wild-type (WT) littermates. Both groups were subjected to a water avoidance stress (WAS) test. Eight weeks after the WAS test, 25% of cchcr1−/− mice exhibited non-inflammatory foci of alopecia on the dorsal skin. On the other hand, none of wild-type littermates cause hair loss. The foci resembled human AA in terms of gross morphology, trichoscopic findings and histological findings. Additionally, gene expression microarray analysis of cchcr1−/− mice revealed abnormalities of hair related genes compared to the control. Our results strongly suggest that CCHCR1 is associated with AA pathogenesis and that cchcr1−/− mice are a good model for investigating AA.

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