scholarly journals Dysregulation of Microtubule Nucleating Proteins in Cancer Cells

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5638
Author(s):  
Pavel Dráber ◽  
Eduarda Dráberová
Keyword(s):  
Cancer Cells ◽  
Cell Behavior ◽  
Microtubule Nucleation ◽  
Microtubule Targeting Agents ◽  
High Resolution Structure ◽  
Ring Complex ◽  
New Compounds ◽  
Centrosomal Proteins ◽  
Insight Into ◽  
Resolution Structure

In cells, microtubules typically nucleate from microtubule organizing centers, such as centrosomes. γ-Tubulin, which forms multiprotein complexes, is essential for nucleation. The γ-tubulin ring complex (γ-TuRC) is an efficient microtubule nucleator that requires additional centrosomal proteins for its activation and targeting. Evidence suggests that there is a dysfunction of centrosomal microtubule nucleation in cancer cells. Despite decades of molecular analysis of γ-TuRC and its interacting factors, the mechanisms of microtubule nucleation in normal and cancer cells remains obscure. Here, we review recent work on the high-resolution structure of γ-TuRC, which brings new insight into the mechanism of microtubule nucleation. We discuss the effects of γ-TuRC protein dysregulation on cancer cell behavior and new compounds targeting γ-tubulin. Drugs inhibiting γ-TuRC functions could represent an alternative to microtubule targeting agents in cancer chemotherapy.

scholarly journals Characterization and Reconstitution of Drosophila γ-Tubulin Ring Complex Subunits

2000 ◽  
Vol 151 (7) ◽  
pp. 1513-1524 ◽  
Author(s):  
Ruwanthi N. Gunawardane ◽  
Ona C. Martin ◽  
Kan Cao ◽  
Lijun Zhang ◽  
Kimberly Dej ◽  
...  
Keyword(s):  
Expression System ◽  
Baculovirus Expression System ◽  
Sequence Motifs ◽  
Microtubule Nucleation ◽  
Sequence Comparisons ◽  
Baculovirus Expression ◽  
Ring Complex ◽  
And Function ◽  
First Time ◽  
Centrosomal Proteins

The γ-tubulin ring complex (γTuRC) is important for microtubule nucleation from the centrosome. In addition to γ-tubulin, the Drosophila γTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small γ-tubulin complex (γTuSC) and the γTuRC, while the remaining subunits are found only in the γTuRC. To study γTuRC assembly and function, we first reconstituted γTuSC using the baculovirus expression system. Using the reconstituted γTuSC, we showed for the first time that this subcomplex of the γTuRC has microtubule binding and capping activities. Next, we characterized two new γTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known γTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with γTuSC. However, this interaction is insufficient for γTuRC assembly.

scholarly journals Recruitment of the γ-Tubulin Ring Complex to Drosophila Salt-stripped Centrosome Scaffolds

1998 ◽  
Vol 142 (3) ◽  
pp. 775-786 ◽  
Author(s):  
Michelle Moritz ◽  
Yixian Zheng ◽  
Bruce M. Alberts ◽  
Karen Oegema
Keyword(s):  
Soluble Fraction ◽  
Protein Complexes ◽  
Drosophila Embryo ◽  
Microtubule Nucleation ◽  
Complementation Assay ◽  
Embryo Extract ◽  
Ring Complex ◽  
Centrosomal Proteins

Extracting isolated Drosophila centrosomes with 2 M KI generates salt-resistant scaffolds that lack the centrosomal proteins CP190, CP60, centrosomin, and γ-tubulin. To clarify the role of these proteins in microtubule nucleation by centrosomes and to identify additional centrosome components required for nucleation, we have developed an in vitro complementation assay for centrosome function. Centrosome aster formation is reconstituted when these inactive, salt-stripped centrosome scaffolds are supplemented with a soluble fraction of a Drosophila embryo extract. The CP60 and CP190 can be removed from this extract without effect, whereas removing the γ-tubulin destroys the complementing activity. Consistent with these results, we find no evidence that these three proteins form a complex together. Instead, γ-tubulin is found in two distinct protein complexes of 240,000 and ∼3,000,000 D. The larger complex, which is analogous to the Xenopus γ-tubulin ring complex (γTuRC) (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578–583), is necessary but not sufficient for complementation. An additional factor found in the extract is required. These results provide the first evidence that the γTuRC is required for microtubule nucleation at the centrosome.

scholarly journals Nanofilaments on glioblastoma exosomes revealed by peak force microscopy

2014 ◽  
Vol 11 (92) ◽  
pp. 20131150 ◽  
Author(s):  
Shivani Sharma ◽  
Kingshuk Das ◽  
JungReem Woo ◽  
James K. Gimzewski
Keyword(s):  
High Resolution ◽  
Cancer Cells ◽  
Extracellular Vesicles ◽  
Intercellular Communication ◽  
Peak Force ◽  
Structural Knowledge ◽  
Force Microscopy ◽  
High Resolution Structure ◽  
Resolution Structure

Exosomes are sub-100 nm extracellular vesicles secreted by normal and cancer cells. We present a high-resolution structure of previously unidentified nanofilaments on glioblastoma-derived exosomes, using nanoscale peak force imaging. These stiff, adhesive, trypsin- and RNAse-resistant surface nanofilaments add a new dimension to the current structural knowledge of exosome-mediated intercellular communication.

scholarly journals Structure of a germline-like human antibody defines a neutralizing epitope on the SARS-CoV-2 spike NTD

2021 ◽  
Author(s):  
Clara Gilda Altomare ◽  
Daniel Cole Adelsberg ◽  
Juan Manuel Carreno ◽  
Iden Avery Sapse ◽  
Fatima Amanat ◽  
...  
Keyword(s):  
Arms Race ◽  
Human Antibody ◽  
Neutralizing Epitope ◽  
Vaccine Immunogen ◽  
Direct Competition ◽  
High Resolution Structure ◽  
Immunogen Design ◽  
Insight Into ◽  
Resolution Structure

Structural characterization of infection- and vaccination-elicited antibodies in complex with antigen provides insight into the evolutionary arms race between the host and the pathogen and informs rational vaccine immunogen design. We isolated a germline-like monoclonal antibody (mAb) from plasmablasts activated upon mRNA vaccination against SARS-CoV-2 and determined its structure in complex with the spike glycoprotein by cryo-EM. We show that the mAb engages a previously uncharacterized neutralizing epitope on the spike N-terminal domain (NTD). The high-resolution structure reveals details of the intermolecular interactions and shows that the mAb inserts its HCDR3 loop into a hydrophobic NTD cavity previously shown to bind a heme metabolite, biliverdin. We demonstrate direct competition with biliverdin and that - because of the conserved nature of the epitope - the mAb maintains binding to viral variants B.1.1.7 and B.1.351. Our study illustrates the feasibility of targeting the NTD to achieve broad neutralization against SARS-CoV-2 variants.

scholarly journals A Pyranoxanthone as a Potent Antimitotic and Sensitizer of Cancer Cells to Low Doses of Paclitaxel

Molecules ◽  
2020 ◽  
Vol 25 (24) ◽  
pp. 5845
Author(s):  
Fábio França ◽  
Patrícia M. A. Silva ◽  
José X. Soares ◽  
Ana C. Henriques ◽  
Daniela R. P. Loureiro ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Arrest ◽  
Antimitotic Activity ◽  
Chromosome Congression ◽  
Antitumor Potential ◽  
Microtubule Targeting Agents ◽  
Growth Activity ◽  
New Compounds ◽  
Combination Regimens

Microtubule-targeting agents (MTAs) remain a gold standard for the treatment of several cancer types. By interfering with microtubules dynamic, MTAs induce a mitotic arrest followed by cell death. This antimitotic activity of MTAs is dependent on the spindle assembly checkpoint (SAC), which monitors the integrity of the mitotic spindle and proper chromosome attachments to microtubules in order to ensure accurate chromosome segregation and timely anaphase onset. However, the cytotoxic activity of MTAs is restrained by drug resistance and/or toxicities, and had motivated the search for new compounds and/or alternative therapeutic strategies. Here, we describe the synthesis and mechanism of action of the xanthone derivative pyranoxanthone 2 that exhibits a potent anti-growth activity against cancer cells. We found that cancer cells treated with the pyranoxanthone 2 exhibited persistent defects in chromosome congression during mitosis that were not corrected over time, which induced a prolonged SAC-dependent mitotic arrest followed by massive apoptosis. Importantly, pyranoxanthone 2 was able to potentiate apoptosis of cancer cells treated with nanomolar concentrations of paclitaxel. Our data identified the potential of the pyranoxanthone 2 as a new potent antimitotic with promising antitumor potential, either alone or in combination regimens.

A High-Resolution Structure that Provides Insight into Coiled-Coil Thiodepsipeptide Dynamic Chemistry

2013 ◽  
Vol 52 (38) ◽  
pp. 9944-9947 ◽  
Author(s):  
Zehavit Dadon ◽  
Manickasundaram Samiappan ◽  
Anat Shahar ◽  
Raz Zarivach ◽  
Gonen Ashkenasy
Keyword(s):  
High Resolution ◽  
Coiled Coil ◽  
High Resolution Structure ◽  
Insight Into ◽  
Resolution Structure

scholarly journals Structure of papain-like protease from SARS-CoV-2 and its complexes with non-covalent inhibitors

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jerzy Osipiuk ◽  
Saara-Anne Azizi ◽  
Steve Dvorkin ◽  
Michael Endres ◽  
Robert Jedrzejczak ◽  
...  
Keyword(s):  
Host Responses ◽  
Peptidase Activity ◽  
Clinical Value ◽  
Structure Based Drug Design ◽  
Replication Studies ◽  
Covalent Inhibitors ◽  
High Resolution Structure ◽  
Insight Into ◽  
Resolution Structure

AbstractThe pandemic caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to expand. Papain-like protease (PLpro) is one of two SARS-CoV-2 proteases potentially targetable with antivirals. PLpro is an attractive target because it plays an essential role in cleavage and maturation of viral polyproteins, assembly of the replicase-transcriptase complex, and disruption of host responses. We report a substantive body of structural, biochemical, and virus replication studies that identify several inhibitors of the SARS-CoV-2 enzyme. We determined the high resolution structure of wild-type PLpro, the active site C111S mutant, and their complexes with inhibitors. This collection of structures details inhibitors recognition and interactions providing fundamental molecular and mechanistic insight into PLpro. All compounds inhibit the peptidase activity of PLpro in vitro, some block SARS-CoV-2 replication in cell culture assays. These findings will accelerate structure-based drug design efforts targeting PLpro to identify high-affinity inhibitors of clinical value.

A High-Resolution Structure that Provides Insight into Coiled-Coil Thiodepsipeptide Dynamic Chemistry

2013 ◽  
Vol 125 (38) ◽  
pp. 10128-10131 ◽  
Author(s):  
Zehavit Dadon ◽  
Manickasundaram Samiappan ◽  
Anat Shahar ◽  
Raz Zarivach ◽  
Gonen Ashkenasy
Keyword(s):  
High Resolution ◽  
Coiled Coil ◽  
High Resolution Structure ◽  
Insight Into ◽  
Resolution Structure

scholarly journals High-Resolution Structure of the Nitrile Reductase QueF Combined with Molecular Simulations Provide Insight into Enzyme Mechanism

2010 ◽  
Vol 404 (1) ◽  
pp. 127-137 ◽  
Author(s):  
Youngchang Kim ◽  
Min Zhou ◽  
Shiu Moy ◽  
Jennifer Morales ◽  
Mark A. Cunningham ◽  
...  
Keyword(s):  
High Resolution ◽  
Molecular Simulations ◽  
Enzyme Mechanism ◽  
High Resolution Structure ◽  
Insight Into ◽  
Resolution Structure

High-resolution structure determination by ALCHEMI

1983 ◽  
Vol 41 ◽  
pp. 178-181 ◽  
Author(s):  
Peter G. Self ◽  
Peter R. Buseck
Keyword(s):  
Site Occupancy ◽  
Real Space ◽  
Unit Cell ◽  
Bragg Angle ◽  
Electron Current ◽  
High Resolution Structure ◽  
Beam Incident ◽  
Crystallographic Planes ◽  
Electron Beam Incident ◽  
Resolution Structure

ALCHEMI (Atom Location by CHanneling Enhanced Microanalysis) enables the site occupancy of atoms in single crystals to be determined. In this article the fundamentals of the method for both EDS and EELS will be discussed. Unlike HRTEM, ALCHEMI does not place stringent resolution requirements on the microscope and, because EDS clearly distinguishes between elements of similar atomic number, it can offer some advantages over HRTEM. It does however, place certain constraints on the crystal. These constraints are: a) the sites of interest must lie on alternate crystallographic planes, b) the projected charge density on the alternate planes must be significantly different, and c) there must be at least one atomic species that lies solely on one of the planes.An electron beam incident on a crystal undergoes elastic scattering; in reciprocal space this is seen as a diffraction pattern and in real space this is a modulation of the electron current across the unit cell. When diffraction is strong (i.e., when the crystal is oriented near to the Bragg angle of a low-order reflection) the electron current at one point in the unit cell will differ significantly from that at another point.

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