Aurora A activation in mitosis promoted by BuGZ

Protein phase separation or coacervation has emerged as a potential mechanism to regulate biological functions. We have shown that coacervation of a mostly unstructured protein, BuGZ, promotes assembly of spindle and its matrix. BuGZ in the spindle matrix binds and concentrates tubulin to promote microtubule (MT) assembly. It remains unclear, however, whether BuGZ could regulate additional proteins to promote spindle assembly. In this study, we report that BuGZ promotes Aurora A (AurA) activation in vitro. Depletion of BuGZ in cells reduces the amount of phosphorylated AurA on spindle MTs. BuGZ also enhances MCAK phosphorylation. The two zinc fingers in BuGZ directly bind to the kinase domain of AurA, which allows AurA to incorporate into the coacervates formed by BuGZ in vitro. Importantly, mutant BuGZ that disrupts the coacervation activity in vitro fails to promote AurA phosphorylation in Xenopus laevis egg extracts. These results suggest that BuGZ coacervation promotes AurA activation in mitosis.

Download Full-text

MicroRNAs in Female Malignancies

Cancer Informatics ◽

10.1177/1176935119828746 ◽

2019 ◽

Vol 18 ◽

pp. 117693511982874 ◽

Cited By ~ 3

Author(s):

Themis Liolios ◽

Stavroula Lila Kastora ◽

Giorgia Colombo

Keyword(s):

Gene Expression ◽

Computational Analysis ◽

Target Genes ◽

Vulvar Cancer ◽

Chemotherapeutic Agents ◽

In Vitro Assays ◽

Biological Processes ◽

Potential Mechanism ◽

Biological Functions

MicroRNAs (miRNAs) are endogenous 22-nucleotide RNAs that can play a fundamental regulatory role in the gene expression of various organisms. Current research suggests that miRNAs can assume pivotal roles in carcinogenesis. In this article, through bioinformatics mining and computational analysis, we determine a single miRNA commonly involved in the development of breast, cervical, endometrial, ovarian, and vulvar cancer, whereas we underline the existence of 7 more miRNAs common in all examined malignancies with the exception of vulvar cancer. Furthermore, we identify their target genes and encoded biological functions. We also analyze common biological processes on which all of the identified miRNAs act and we suggest a potential mechanism of action. In addition, we analyze exclusive miRNAs among the examined malignancies and bioinformatically explore their functionality. Collectively, our data can be employed in in vitro assays as a stepping stone in the identification of a universal machinery that is derailed in female malignancies, whereas exclusive miRNAs may be employed as putative targets for future chemotherapeutic agents or cancer-specific biomarkers.

Download Full-text

Phase separation of BuGZ promotes Aurora A activation and spindle assembly

The Journal of Cell Biology ◽

10.1083/jcb.201711070 ◽

2017 ◽

Vol 217 (1) ◽

pp. 09-10 ◽

Cited By ~ 1

Author(s):

Jeffrey B. Woodruff

Keyword(s):

Phase Separation ◽

Mitotic Spindle ◽

Matrix Protein ◽

Spindle Assembly ◽

Aurora A ◽

Spindle Matrix ◽

Cell Biol ◽

Mitotic Spindle Assembly

The spindle matrix has been proposed to facilitate mitotic spindle assembly. In this issue, Huang et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201706103) show that the spindle matrix protein BuGZ is sufficient to form micron-scale compartments that recruit and activate Aurora A, a critical kinase for spindle assembly.

Download Full-text

Bora phosphorylation substitutes in trans for T-loop phosphorylation in Aurora A to promote mitotic entry

Nature Communications ◽

10.1038/s41467-021-21922-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

N. Tavernier ◽

Y. Thomas ◽

S. Vigneron ◽

P. Maisonneuve ◽

S. Orlicky ◽

...

Keyword(s):

Kinase Domain ◽

Activation Mechanism ◽

Aurora A Kinase ◽

Aurora A ◽

Mitotic Entry ◽

Amino Acid Region ◽

Egg Extracts ◽

Activation Segment ◽

Xenopus Egg ◽

Xenopus Egg Extracts

AbstractPolo-like kinase 1 (Plk1) is instrumental for mitotic entry and progression. Plk1 is activated by phosphorylation on a conserved residue Thr210 in its activation segment by the Aurora A kinase (AURKA), a reaction that critically requires the co-factor Bora phosphorylated by a CyclinA/B-Cdk1 kinase. Here we show that phospho-Bora is a direct activator of AURKA kinase activity. We localize the key determinants of phospho-Bora function to a 100 amino acid region encompassing two short Tpx2-like motifs and a phosphoSerine-Proline motif at Serine 112, through which Bora binds AURKA. The latter substitutes in trans for the Thr288 phospho-regulatory site of AURKA, which is essential for an active conformation of the kinase domain. We demonstrate the importance of these determinants for Bora function in mitotic entry both in Xenopus egg extracts and in human cells. Our findings unveil the activation mechanism of AURKA that is critical for mitotic entry.

Download Full-text

Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis

The Journal of Cell Biology ◽

10.1083/jcb.200503023 ◽

2005 ◽

Vol 170 (7) ◽

pp. 1047-1055 ◽

Cited By ~ 166

Author(s):

Kazuhisa Kinoshita ◽

Tim L. Noetzel ◽

Laurence Pelletier ◽

Karl Mechtler ◽

David N. Drechsel ◽

...

Keyword(s):

Cell Cycle ◽

Xenopus Laevis ◽

Microtubule Assembly ◽

Aurora A ◽

Specific Mechanism ◽

Microtubule Polymerization ◽

Egg Extracts ◽

Microtubule Growth

Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3–XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A–dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.

Download Full-text

Mechanistic Studies of the Mitotic Activation of Mos

Molecular and Cellular Biology ◽

10.1128/mcb.00273-06 ◽

2006 ◽

Vol 26 (14) ◽

pp. 5300-5309 ◽

Cited By ~ 13

Author(s):

Jianbo Yue ◽

James E. Ferrell

Keyword(s):

Protein Kinase ◽

Oocyte Maturation ◽

Tyrosine Kinases ◽

Spindle Assembly Checkpoint ◽

Phosphorylation Site ◽

Kinase Domain ◽

Mitogen Activated Protein Kinase ◽

Egg Extracts ◽

Mitogen Activated Protein

ABSTRACT The protein kinase Mos is responsible for the activation of MEK1 and p42 mitogen-activated protein kinase during Xenopus oocyte maturation and during mitosis in Xenopus egg extracts. Here we show that the activation of Mos depends upon the phosphorylation of Ser 3, a residue previously implicated in the regulation of Mos stability; the dephosphorylation of Ser 105, a previously unidentified phosphorylation site conserved in Mos proteins; and the regulated dissociation of Mos from CK2β. Mutation of Ser 3 to alanine and/or mutation of Ser 105 to glutamate produces a Mos protein that is defective for M-phase activation, as assessed by in vitro kinase assays, and defective for induction of oocyte maturation and maintenance of the spindle assembly checkpoint in extracts. Interestingly, Ser 105 is situated at the beginning of helix αC in the N-terminal lobe of the Mos kinase domain. Changes in the orientation of this helix have been previously implicated in the activation of Cdk2 and Src family tyrosine kinases. Our work suggests that Ser 105 dephosphorylation represents a novel mechanism for reorienting helix αC.

Download Full-text

FORMULATION AND IN VITRO, IN VIVO EVALUATION OF PRONIOSOMAL GEL OF NEOMYCIN SULPHATE

International Journal of Applied Pharmaceutics ◽

10.22159/ijap.2019v11i2.30614 ◽

2019 ◽

pp. 156-163

Author(s):

AMOL SHETE ◽

PRIYANKA THORAT ◽

RAJENDRA DOIJAD ◽

SACHIN SAJANE

Keyword(s):

Phase Separation ◽

Skin Irritation ◽

Entrapment Efficiency ◽

Separation Method ◽

Gelling Agent ◽

In Vivo Evaluation ◽

Skin Delivery ◽

Span 60

Objective: The objectives of present investigation were to prepare and evaluate proniosomes of neomycin sulphate (NS) by coacervation phase separation method by using sorbitan monostearate (span 60) and lecithin as a surfactant to increase the penetration through the skin and study the effect of concentration of the same. Methods: Proniosomes of neomycin sulphate (NS) were prepared by coacervation phase separation method by using span 60 and lecithin. The effect of concentration of span 60 and lecithin was studied by factorial design. The prepared proniosomes were converted to gel by using carbopol as a gelling agent. The prepared formulations were evaluated for entrapment efficiency, in vitro drug diffusion, in vitro antibacterial activity and in vivo skin irritation test etc. Results: All Formulation showed the percentage entrapment efficiency in the range 38.31±0.05% to 77.96±0.06%, good homogeneity and gel was easily spreadable with minimal of shear. Optimized formulation showed enhanced rate of diffusion in vitro, increase in zone of inhibition against staphylococcus aureus, no skin irritation and showed good stability. Conclusion: The results of present study indicates that proniosomal gel formulated by using combination of span 60, Lecithin, cholesterol can be used to enhance skin delivery of NS because of excellent permeation of drug. Developed proniosomal gel formulation was promising carrier for NS

Download Full-text

Molecular Insights into an Antibiotic Enhancer Action of New Morpholine-Containing 5-Arylideneimidazolones in the Fight against MDR Bacteria

International Journal of Molecular Sciences ◽

10.3390/ijms22042062 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2062

Author(s):

Aneta Kaczor ◽

Karolina Witek ◽

Sabina Podlewska ◽

Veronique Sinou ◽

Joanna Czekajewska ◽

...

Keyword(s):

Molecular Modelling ◽

Efflux Pump ◽

Gram Negative Bacteria ◽

Potential Mechanism ◽

Multidrug Efflux ◽

Aromatic Rings ◽

Allosteric Site ◽

Multidrug Efflux Pump ◽

Dual Action

In the search for an effective strategy to overcome antimicrobial resistance, a series of new morpholine-containing 5-arylideneimidazolones differing within either the amine moiety or at position five of imidazolones was explored as potential antibiotic adjuvants against Gram-positive and Gram-negative bacteria. Compounds (7–23) were tested for oxacillin adjuvant properties in the Methicillin-susceptible S. aureus (MSSA) strain ATCC 25923 and Methicillin-resistant S. aureus MRSA 19449. Compounds 14–16 were tested additionally in combination with various antibiotics. Molecular modelling was performed to assess potential mechanism of action. Microdilution and real-time efflux (RTE) assays were carried out in strains of K. aerogenes to determine the potential of compounds 7–23 to block the multidrug efflux pump AcrAB-TolC. Drug-like properties were determined experimentally. Two compounds (10, 15) containing non-condensed aromatic rings, significantly reduced oxacillin MICs in MRSA 19449, while 15 additionally enhanced the effectiveness of ampicillin. Results of molecular modelling confirmed the interaction with the allosteric site of PBP2a as a probable MDR-reversing mechanism. In RTE, the compounds inhibited AcrAB-TolC even to 90% (19). The 4-phenylbenzylidene derivative (15) demonstrated significant MDR-reversal “dual action” for β-lactam antibiotics in MRSA and inhibited AcrAB-TolC in K. aerogenes. 15 displayed also satisfied solubility and safety towards CYP3A4 in vitro.

Download Full-text

Solvent Exposure and Ionic Condensation Drive Fuzzy Dimerization of Disordered Heterochromatin Protein Sequence

Biomolecules ◽

10.3390/biom11060915 ◽

2021 ◽

Vol 11 (6) ◽

pp. 915

Author(s):

Jazelli Mueterthies ◽

Davit A. Potoyan

Keyword(s):

Phase Separation ◽

Low Complexity ◽

Nuclear Bodies ◽

Disordered Proteins ◽

Solvent Exposure ◽

Ion Release ◽

Dimer Formation ◽

Eukaryotic Organisms

Proteins with low complexity, disordered sequences are receiving increasing attention due to their central roles in the biogenesis and regulation of membraneless organelles. In eukaryotic organisms, a substantial fraction of disordered proteins reside in the nucleus, thereby facilitating the formation of nuclear bodies, nucleolus, and chromatin compartmentalization. The heterochromatin family of proteins (HP1) is an important player in driving the formation of gene silenced mesoscopic heterochromatin B compartments and pericentric regions. Recent experiments have shown that the HP1a sequence of Drosophila melanogaster can undergo liquid-liquid phase separation under both in vitro and in vivo conditions, induced by changes of the monovalent salt concentration. While the phase separation of HP1a is thought to be the mechanism underlying chromatin compartmentalization, the molecular level mechanistic picture of salt-driven phase separation of HP1a has remained poorly understood. The disordered hinge region of HP1a is seen as the driver of salt-induced condensation because of its charge enriched sequence and post-translational modifications. Here, we set out to decipher the mechanisms of salt-induced condensation of HP1a through a systematic study of salt-dependent conformations of single chains and fuzzy dimers of disordered HP1a hinge sequences. Using multiple independent all-atom simulations with and without enhanced sampling, we carry out detailed characterization of conformational ensembles of disordered HP1a chains under different ionic conditions using various polymeric and structural measures. We show that the mobile ion release, enhancement of local transient secondary structural elements, and side-chain exposure to solvent are robust trends that accompany fuzzy dimer formation. Furthermore, we find that salt-induced changes in the ensemble of conformations of HP1a disordered hinge sequence fine-tune the inter-chain vs. self-chain interactions in ways that favor fuzzy dimer formation under low salt conditions in the agreement with condensation trends seen in experiments.

Download Full-text

Targeting NSD2-mediated SRC-3 liquid–liquid phase separation sensitizes bortezomib treatment in multiple myeloma

Nature Communications ◽

10.1038/s41467-021-21386-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jing Liu ◽

Ying Xie ◽

Jing Guo ◽

Xin Li ◽

Jingjing Wang ◽

...

Keyword(s):

Multiple Myeloma ◽

Drug Resistance ◽

Phase Separation ◽

Liquid Phase ◽

Liquid Phase Separation ◽

Acquired Drug Resistance ◽

Bortezomib Treatment ◽

Liquid Liquid Phase Separation

AbstractDevelopment of chemoresistance is the main reason for failure of clinical management of multiple myeloma (MM), but the genetic and epigenetic aberrations that interact to confer such chemoresistance remains unknown. In the present study, we find that high steroid receptor coactivator-3 (SRC-3) expression is correlated with relapse/refractory and poor outcomes in MM patients treated with bortezomib (BTZ)-based regimens. Furthermore, in immortalized cell lines, high SRC-3 enhances resistance to proteasome inhibitor (PI)-induced apoptosis. Overexpressed histone methyltransferase NSD2 in patients bearing a t(4;14) translocation or in BTZ-resistant MM cells coordinates elevated SRC-3 by enhancing its liquid–liquid phase separation to supranormally modify histone H3 lysine 36 dimethylation (H3K36me2) modifications on promoters of anti-apoptotic genes. Targeting SRC-3 or interference of its interactions with NSD2 using a newly developed inhibitor, SI-2, sensitizes BTZ treatment and overcomes drug resistance both in vitro and in vivo. Taken together, our findings elucidate a previously unrecognized orchestration of SRC-3 and NSD2 in acquired drug resistance of MM and suggest that SI-2 may be efficacious for overcoming drug resistance in MM patients.

Download Full-text

E3 ligase Nedd4l promotes antiviral innate immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3

Nature Communications ◽

10.1038/s41467-021-21456-1 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Peng Gao ◽

Xianwei Ma ◽

Ming Yuan ◽

Yulan Yi ◽

Guoke Liu ◽

...

Keyword(s):

Posttranslational Modifications ◽

Type I Interferon ◽

Zinc Fingers ◽

E3 Ligase ◽

Type I ◽

E3 Ligases ◽

Protein Posttranslational Modifications ◽

Antiviral Innate Immunity

AbstractUbiquitination is one of the most prevalent protein posttranslational modifications. Here, we show that E3 ligase Nedd4l positively regulates antiviral immunity by catalyzing K29-linked cysteine ubiquitination of TRAF3. Deficiency of Nedd4l significantly impairs type I interferon and proinflammatory cytokine production induced by virus infection both in vitro and in vivo. Nedd4l deficiency inhibits virus-induced ubiquitination of TRAF3, the binding between TRAF3 and TBK1, and subsequent phosphorylation of TBK1 and IRF3. Nedd4l directly interacts with TRAF3 and catalyzes K29-linked ubiquitination of Cys56 and Cys124, two cysteines that constitute zinc fingers, resulting in enhanced association between TRAF3 and E3 ligases, cIAP1/2 and HECTD3, and also increased K48/K63-linked ubiquitination of TRAF3. Mutation of Cys56 and Cys124 diminishes Nedd4l-catalyzed K29-linked ubiquitination, but enhances association between TRAF3 and the E3 ligases, supporting Nedd4l promotes type I interferon production in response to virus by catalyzing ubiquitination of the cysteines in TRAF3.

Download Full-text