Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation.

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Identification of ARKL1 as a Negative Regulator of Epstein-Barr Virus Reactivation.

J Virol. 2019 Jul 24;:

Authors: Siddiqi UZ, Vaidya AS, Li X, Marcon E, Tsao SW, Greenblatt J, Frappier L

Abstract
Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication. Lytic reactivation starts with derepression of the Zp promoter controlling BZLF1 gene expression, which binds and is activated by the c-Jun transcriptional activator. Here we identified the cellular Arkadia-like 1 (ARKL1) protein as a negative regulator of Zp and EBV reactivation. Silencing of ARKL1 in the context of EBV-positive gastric carcinoma (AGS), nasopharyngeal carcinoma (NPC43) and B cells (M81) led to increased lytic protein expression, whereas overexpression inhibited BZLF1 expression. Similar effects of ARKL1 modulation were seen on BZLF1 transcripts as well as on Zp activity in Zp reporter assays, showing ARKL1 repressed Zp. Proteomic profiling of ARKL1-host interactions identified c-Jun as an ARKL1 interactor, and reporter assays for Jun transcriptional activity showed that ARKL1 inhibited Jun activity. The ARKL1-Jun interaction required ARKL1 sequences that we previously showed mediated binding to the CK2 kinase regulatory subunit, CK2β, suggesting that CK2β might mediate the ARKL1-Jun interaction. This model was supported by the findings that silencing of CK2β, but not the CK2α catalytic subunit, abrogated the ARKL1-Jun interaction and phenocopied ARKL1 silencing in promoting EBV reactivation. Additionally, ARKL1 associated with Zp in reporter assays and this was increased by additional CK2β. Together the data indicate that ARKL1 is a negative regulator of Zp and EBV reactivation that acts by inhibiting Jun activity through a CK2β-mediated interaction.IMPORTANCE Epstein-Barr virus (EBV) maintains a life-long infection due to the ability to alternate between latent and lytic modes of replication and is associated with several types of cancer. We have identified a cellular protein (ARKL1) that acts to repress the reactivation of EBV from the latent to the lytic cycle. We show that ARKL1 acts to repress transcription of the EBV lytic switch protein by inhibiting the activity of the cellular transcription factor, c-Jun. This not only provides a new mechanism of regulating EBV reactivation but also identifies a novel cellular function of ARKL1 as an inhibitor of Jun-mediated transcription.

PMID: 31341047 [PubMed - as supplied by publisher]



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Benchmarking to the Gold Standard: Hyaluronan-Oxime Hydrogels Recapitulate Xenograft Models with In Vitro Breast Cancer Spheroid Culture.

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Benchmarking to the Gold Standard: Hyaluronan-Oxime Hydrogels Recapitulate Xenograft Models with In Vitro Breast Cancer Spheroid Culture.

Adv Mater. 2019 Jul 19;:e1901166

Authors: Baker AEG, Bahlmann LC, Tam RY, Liu JC, Ganesh AN, Mitrousis N, Marcellus R, Spears M, Bartlett JMS, Cescon DW, Bader GD, Shoichet MS

Abstract
Many 3D in vitro models induce breast cancer spheroid formation; however, this alone does not recapitulate the complex in vivo phenotype. To effectively screen therapeutics, it is urgently needed to validate in vitro cancer spheroid models against the gold standard of xenografts. A new oxime-crosslinked hyaluronan (HA) hydrogel is designed, manipulating gelation rate and mechanical properties to grow breast cancer spheroids in 3D. This HA-oxime breast cancer model maintains the gene expression profile most similar to that of tumor xenografts based on a pan-cancer gene expression profile (comprising 730 genes) of three different human breast cancer subtypes compared to Matrigel or conventional 2D culture. Differences in gene expression between breast cancer cultures in HA-oxime versus Matrigel or 2D are confirmed for 12 canonical pathways by gene set variation analysis. Importantly, drug response is dependent on the culture method. Breast cancer cells respond better to the Rac inhibitor (EHT-1864) and the PI3K inhibitor (AZD6482) when cultured in HA-oxime versus Matrigel. This study demonstrates the superiority of an HA-based hydrogel as a platform for in vitro breast cancer culture of both primary, patient-derived cells and cell lines, and provides a hydrogel culture model that closely matches that in vivo.

PMID: 31322299 [PubMed - as supplied by publisher]



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EPIC: software toolkit for elution profile-based inference of protein complexes.

EPIC: software toolkit for elution profile-based inference of protein complexes.

Nat Methods. 2019 Jul 15;:

Authors: Hu LZ, Goebels F, Tan JH, Wolf E, Kuzmanov U, Wan C, Phanse S, Xu C, Schertzberg M, Fraser AG, Bader GD, Emili A

Abstract
Protein complexes are key macromolecular machines of the cell, but their description remains incomplete. We and others previously reported an experimental strategy for global characterization of native protein assemblies based on chromatographic fractionation of biological extracts coupled to precision mass spectrometry analysis (chromatographic fractionation-mass spectrometry, CF-MS), but the resulting data are challenging to process and interpret. Here, we describe EPIC (elution profile-based inference of complexes), a software toolkit for automated scoring of large-scale CF-MS data to define high-confidence multi-component macromolecules from diverse biological specimens. As a case study, we used EPIC to map the global interactome of Caenorhabditis elegans, defining 612 putative worm protein complexes linked to diverse biological processes. These included novel subunits and assemblies unique to nematodes that we validated using orthogonal methods. The open source EPIC software is freely available as a Jupyter notebook packaged in a Docker container ( https://hub.docker.com/r/baderlab/bio-epic/ ).

PMID: 31308550 [PubMed - as supplied by publisher]



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Identification and Characterization of Mutations in Ubiquitin Required for Non-covalent Dimer Formation.

Identification and Characterization of Mutations in Ubiquitin Required for Non-covalent Dimer Formation.

Structure. 2019 Jul 05;:

Authors: Gabrielsen M, Buetow L, Kowalczyk D, Zhang W, Sidhu SS, Huang DT

Abstract
Ubiquitin (Ub) is a small protein that post-translationally modifies a variety of substrates in eukaryotic cells to modulate substrate function. The ability of Ub to interact with numerous protein domains makes Ub an attractive scaffold for engineering ubiquitin variants (UbVs) with high target specificity. Previously, we identified a UbV that formed a non-covalent stable dimer via a β-strand exchange, and in the current work we identified and characterized the minimal substitutions in the primary sequence of Ub required to form a higher ordered complex. Using solution angle scattering and X-ray crystallography, we show that a single substitution of residue Gly10 to either Ala or Val is sufficient to convert Ub from a monomer to a dimer. We also investigate contributions to dimer formation by the residues in the surrounding sequence. These results can be used to develop next-generation phage-display libraries of UbVs to engineer new interfaces for protein recognition.

PMID: 31303481 [PubMed - as supplied by publisher]



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Specifications of Standards in Systems and Synthetic Biology: Status and Developments in 2019.

Specifications of Standards in Systems and Synthetic Biology: Status and Developments in 2019.

J Integr Bioinform. 2019 Jul 13;:

Authors: Schreiber F, Sommer B, Bader GD, Gleeson P, Golebiewski M, Hucka M, Keating SM, König M, Myers C, Nickerson D, Waltemath D

Abstract
This special issue of the Journal of Integrative Bioinformatics presents an overview of COMBINE standards and their latest specifications. The standards cover representation formats for computational modeling in synthetic and systems biology and include BioPAX, CellML, NeuroML, SBML, SBGN, SBOL and SED-ML. The articles in this issue contain updated specifications of SBGN Process Description Level 1 Version 2, SBML Level 3 Core Version 2 Release 2, SBOL Version 2.3.0, and SBOL Visual Version 2.1.

PMID: 31301675 [PubMed - as supplied by publisher]



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The optoelectronic microrobot: A versatile toolbox for micromanipulation.

The optoelectronic microrobot: A versatile toolbox for micromanipulation.

Proc Natl Acad Sci U S A. 2019 Jul 09;:

Authors: Zhang S, Scott EY, Singh J, Chen Y, Zhang Y, Elsayed M, Chamberlain MD, Shakiba N, Adams K, Yu S, Morshead CM, Zandstra PW, Wheeler AR

Abstract
Microrobotics extends the reach of human-controlled machines to submillimeter dimensions. We introduce a microrobot that relies on optoelectronic tweezers (OET) that is straightforward to manufacture, can take nearly any desirable shape or form, and can be programmed to carry out sophisticated, multiaxis operations. One particularly useful program is a serial combination of "load," "transport," and "deliver," which can be applied to manipulate a wide range of micrometer-dimension payloads. Importantly, microrobots programmed in this manner are much gentler on fragile mammalian cells than conventional OET techniques. The microrobotic system described here was demonstrated to be useful for single-cell isolation, clonal expansion, RNA sequencing, manipulation within enclosed systems, controlling cell-cell interactions, and isolating precious microtissues from heterogeneous mixtures. We propose that the optoelectronic microrobotic system, which can be implemented using a microscope and consumer-grade optical projector, will be useful for a wide range of applications in the life sciences and beyond.

PMID: 31289234 [PubMed - as supplied by publisher]



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Assessing micrometastases as a target for nanoparticles using 3D microscopy and machine learning.

Assessing micrometastases as a target for nanoparticles using 3D microscopy and machine learning.

Proc Natl Acad Sci U S A. 2019 Jul 08;:

Authors: Kingston BR, Syed AM, Ngai J, Sindhwani S, Chan WCW

Abstract
Metastasis of solid tumors is a key determinant of cancer patient survival. Targeting micrometastases using nanoparticles could offer a way to stop metastatic tumor growth before it causes excessive patient morbidity. However, nanoparticle delivery to micrometastases is difficult to investigate because micrometastases are small in size and lie deep within tissues. Here, we developed an imaging and image analysis workflow to analyze nanoparticle-cell interactions in metastatic tumors. This technique combines tissue clearing and 3D microscopy with machine learning-based image analysis to assess the physiology of micrometastases with single-cell resolution and quantify the delivery of nanoparticles within them. We show that nanoparticles access a higher proportion of cells in micrometastases (50% nanoparticle-positive cells) compared with primary tumors (17% nanoparticle-positive cells) because they reside close to blood vessels and require a small diffusion distance to reach all tumor cells. Furthermore, the high-throughput nature of our image analysis workflow allowed us to profile the physiology and nanoparticle delivery of 1,301 micrometastases. This enabled us to use machine learning-based modeling to predict nanoparticle delivery to individual micrometastases based on their physiology. Our imaging method allows researchers to measure nanoparticle delivery to micrometastases and highlights an opportunity to target micrometastases with nanoparticles. The development of models to predict nanoparticle delivery based on micrometastasis physiology could enable personalized treatments based on the specific physiology of a patient's micrometastases.

PMID: 31285340 [PubMed - as supplied by publisher]



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Global proteomic analyses define an environmentally contingent Hsp90 interactome and reveal chaperone-dependent regulation of stress granule proteins and the R2TP complex in a fungal pathogen.

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Global proteomic analyses define an environmentally contingent Hsp90 interactome and reveal chaperone-dependent regulation of stress granule proteins and the R2TP complex in a fungal pathogen.

PLoS Biol. 2019 Jul 08;17(7):e3000358

Authors: O'Meara TR, O'Meara MJ, Polvi EJ, Pourhaghighi MR, Liston SD, Lin ZY, Veri AO, Emili A, Gingras AC, Cowen LE

Abstract
Hsp90 is a conserved molecular chaperone that assists in the folding and function of diverse cellular regulators, with a profound impact on biology, disease, and evolution. As a central hub of protein interaction networks, Hsp90 engages with hundreds of protein-protein interactions within eukaryotic cells. These interactions include client proteins, which physically interact with Hsp90 and depend on the chaperone for stability or function, as well as co-chaperones and partner proteins that modulate chaperone function. Currently, there are no methods to accurately predict Hsp90 interactors and there has been considerable network rewiring over evolutionary time, necessitating experimental approaches to define the Hsp90 network in the species of interest. This is a pressing challenge for fungal pathogens, for which Hsp90 is a key regulator of stress tolerance, drug resistance, and virulence traits. To address this challenge, we applied a novel biochemical fractionation and quantitative proteomic approach to examine alterations to the proteome upon perturbation of Hsp90 in a leading human fungal pathogen, Candida albicans. In parallel, we performed affinity purification coupled to mass spectrometry to define physical interacting partners for Hsp90 and the Hsp90 co-chaperones and identified 164 Hsp90-interacting proteins, including 111 that are specific to the pathogen. We performed the first analysis of the Hsp90 interactome upon antifungal drug stress and demonstrated that Hsp90 stabilizes processing body (P-body) and stress granule proteins that contribute to drug tolerance. We also describe novel roles for Hsp90 in regulating posttranslational modification of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) complex and the formation of protein aggregates in response to thermal stress. This study provides a global view of the Hsp90 interactome in a fungal pathogen, demonstrates the dynamic role of Hsp90 in response to environmental perturbations, and highlights a novel connection between Hsp90 and the regulation of mRNA-associated protein granules.

PMID: 31283755 [PubMed - as supplied by publisher]



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Assessing predictions on fitness effects of missense variants in calmodulin.

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Assessing predictions on fitness effects of missense variants in calmodulin.

Hum Mutat. 2019 Jul 08;:

Authors: Zhang J, Kinch LN, Cong Q, Katsonis P, Lichtarge O, Savojardo C, Babbi G, Martelli PL, Capriotti E, Casadio R, Garg A, Pal D, Weile J, Sun S, Verby M, Roth FP, Grishin NV

Abstract
This paper reports the evaluation of predictions for the "CALM1" challenge in the 5th round of the Critical Assessment of Genome Interpretation held in 2018. In the challenge, the participants were asked to predict effects on yeast growth caused by missense variants of human calmodulin, a highly conserved protein in eukaryotic cells sensing calcium concentration. The performance of predictors implementing different algorithms and methods is similar. Most predictors are able to identify the deleterious or benign variants with modest accuracy, with a baseline predictor based purely on sequence conservation slightly outperforming the submitted predictions. Nevertheless, we think that the accuracy of predictions remains far from satisfactory, and the field awaits substantial improvements. The most poorly predicted variants in this round surround functional CALM1 sites that bind calcium or peptide, which suggests that better incorporation of structural analysis may help improve predictions. This article is protected by copyright. All rights reserved.

PMID: 31283071 [PubMed - as supplied by publisher]



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The Med31 Conserved Component of the Divergent Mediator Complex in Tetrahymena thermophila Participates in Developmental Regulation.

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The Med31 Conserved Component of the Divergent Mediator Complex in Tetrahymena thermophila Participates in Developmental Regulation.

Curr Biol. 2019 Jun 28;:

Authors: Garg J, Saettone A, Nabeel-Shah S, Cadorin M, Ponce M, Marquez S, Pu S, Greenblatt J, Lambert JP, Pearlman RE, Fillingham J

Abstract
Mediator is a large protein complex required for basal and regulated expression of most RNA polymerase II (RNAP II)-transcribed genes, in part due to its interaction with and phosphorylation of the conserved C-terminal domain (CTD) of Rpb1 [1, 2]. Mediator has been implicated in many aspects of gene expression including chromatin looping [3], higher-order chromatin folding [4], mRNA processing [5] and export [6], and transcriptional memory [7]. Mediator is thought to have played a major role during eukaryotic diversification [8, 9], although its function remains unknown in evolutionarily deep branching eukaryotes lacking canonical CTD heptad repeats. We used the ciliate protozoan Tetrahymena thermophila as a model organism whose genome encodes a highly divergent Rpb1 lacking canonical CTD heptad repeats. We endogenously tagged the Med31 subunit of the Mediator complex and performed affinity purification coupled with mass spectrometry (AP-MS) to identify Mediator subunits. We found that Med31 physically interacts with a large number of proteins (>20), several of which share similarities to canonical Mediator subunits in yeast and humans as well as Tetrahymena-specific proteins. Furthermore, Med31 ChIP-seq analysis suggested a global role for Mediator in transcription regulation. We demonstrated that MED31 knockdown in growing Tetrahymena results in the ectopic expression of developmental genes important for programmed DNA rearrangements. In addition, indirect immunofluorescence revealed Med31 localization in meiotic micronuclei, implicating Mediator in RNAPII-dependent ncRNA transcription. Our results reveal structural and functional insights and implicate Mediator as an ancient cellular machinery for transcription regulation with a possible involvement in global transcription of ncRNAs.

PMID: 31280994 [PubMed - as supplied by publisher]



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