Donnelly Centre for Cellular and Biomolecular Research

PubMed

Recent Publications

Functional analysis and fine mapping of the 9p22.2 ovarian cancer susceptibility locus.

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Functional analysis and fine mapping of the 9p22.2 ovarian cancer susceptibility locus.

Cancer Res. 2018 Nov 28;:

Authors: Buckley MA, Woods NT, Tyrer JP, Mendoza-Fandiño G, Lawrenson K, Hazelett DJ, Najafabadi HS, Gjyshi A, Carvalho RS, Lyra PC, Coetzee SG, Shen HC, Yang AW, Earp MA, Yoder S, Risch H, Chenevix-Trench G, Ramus SJ, Phelan CM, Coetzee GA, Noushmehr H, Hughes TR, Sellers TA, Goode EL, Pharoah PDP, Gayther SA, Monteiro AN

Abstract
Genome-wide association studies have identified 40 ovarian cancer risk loci. However, the mechanisms underlying these associations remain elusive. In this study, we conducted a two-pronged approach to identify candidate causal SNPs and assess underlying biological mechanisms at chromosome 9p22.2, the first and most statistically significant associated locus for ovarian cancer susceptibility. Three transcriptional regulatory elements with allele-specific effects and a scaffold/matrix attachment region were characterized and through physical DNA interactions BNC2 was established as the most likely target gene. We determined the consensus binding sequence for BNC2 in vitro, verified its enrichment in BNC2 ChIP-Seq regions and validated a set of its downstream target genes. Fine-mapping by dense regional genotyping in over 15,000 ovarian cancer cases and 30,000 controls identified SNPs in the scaffold/matrix attachment region as among the most likely causal variants. This study reveals a comprehensive regulatory landscape at 9p22.2 and proposes a likely mechanism of susceptibility to ovarian cancer.

PMID: 30487138 [PubMed - as supplied by publisher]



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Surface loops in a single SH2 domain are capable of encoding the spectrum of specificity of the SH2 family.

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Surface loops in a single SH2 domain are capable of encoding the spectrum of specificity of the SH2 family.

Mol Cell Proteomics. 2018 Nov 27;:

Authors: Liu H, Huang H, Voss C, Kaneko T, Qin W, Sidhu S, Shun-Cheng Li SS

Abstract
Src homology 2 (SH2) domains play an essential role in cellular signal transduction by binding to proteins phosphorylated on Tyr residue. Although Tyr phosphorylation (pY) is a prerequisite for binding for essentially all SH2 domains characterized to date, different SH2 domains prefer specific sequence motifs C-terminal to the pY residue. Because all SH2 domains adopt the same structural fold, it is not well understood how different SH2 domains have acquired the ability to recognize distinct sequence motifs. We have shown previously that the EF and BG loops that connect the secondary structure elements on an SH2 domain dictates its specificity. In this study, we investigated if these surface loops could be engineered to encode diverse specificities.  By characterizing a group of SH2 variants selected by different pY peptides from phage-displayed libraries, we show that the EF and BG loops of the Fyn SH2 domain are capable of encoding a wide spectrum of specificities, including all three major specificity classes (P+2, P+3 and P+4) of the SH2 domain family. Furthermore, we found that the specificity of a given variant correlates with the sequence feature of the bait peptide used for its isolation, suggesting that an SH2 domain may acquire specificity by co-evolving with its ligand. Intriguingly, we found that the SH2 variants can employ a variety of different mechanisms to confer the same specificity, suggesting the EF and BG loops are highly flexible and adaptable. Our work provides a plausible mechanism for the SH2 domain to acquire the wide spectrum of specificity observed in nature through loop variation with minimal disturbance to the SH2 fold.  It is likely that similar mechanisms may have been employed by other modular interaction domains to generate diversity in specificity.

PMID: 30482845 [PubMed - as supplied by publisher]



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Function, evolution, and structure of J-domain proteins.

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Function, evolution, and structure of J-domain proteins.

Cell Stress Chaperones. 2018 Nov 26;:

Authors: Kampinga HH, Andreasson C, Barducci A, Cheetham ME, Cyr D, Emanuelsson C, Genevaux P, Gestwicki JE, Goloubinoff P, Huerta-Cepas J, Kirstein J, Liberek K, Mayer MP, Nagata K, Nillegoda NB, Pulido P, Ramos C, De Los Rios P, Rospert S, Rosenzweig R, Sahi C, Taipale M, Tomiczek B, Ushioda R, Young JC, Zimmermann R, Zylicz A, Zylicz M, Craig EA, Marszalek J

Abstract
Hsp70 chaperone systems are very versatile machines present in nearly all living organisms and in nearly all intracellular compartments. They function in many fundamental processes through their facilitation of protein (re)folding, trafficking, remodeling, disaggregation, and degradation. Hsp70 machines are regulated by co-chaperones. J-domain containing proteins (JDPs) are the largest family of Hsp70 co-chaperones and play a determining role functionally specifying and directing Hsp70 functions. Many features of JDPs are not understood; however, a number of JDP experts gathered at a recent CSSI-sponsored workshop in Gdansk (Poland) to discuss various aspects of J-domain protein function, evolution, and structure. In this report, we present the main findings and the consensus reached to help direct future developments in the field of Hsp70 research.

PMID: 30478692 [PubMed - as supplied by publisher]



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An ATG16L1-dependent pathway promotes plasma membrane repair and limits Listeria monocytogenes cell-to-cell spread.

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An ATG16L1-dependent pathway promotes plasma membrane repair and limits Listeria monocytogenes cell-to-cell spread.

Nat Microbiol. 2018 Dec;3(12):1472-1485

Authors: Tan JMJ, Mellouk N, Osborne SE, Ammendolia DA, Dyer DN, Li R, Brunen D, van Rijn JM, Huang J, Czuczman MA, Cemma MA, Won AM, Yip CM, Xavier RJ, MacDuff DA, Reggiori F, Debnath J, Yoshimori T, Kim PK, Fairn GD, Coyaud E, Raught B, Muise AM, Higgins DE, Brumell JH

Abstract
Plasma membrane integrity is essential for the viability of eukaryotic cells. In response to bacterial pore-forming toxins, disrupted regions of the membrane are rapidly repaired. However, the pathways that mediate plasma membrane repair are unclear. Here we show that autophagy-related (ATG) protein ATG16L1 and its binding partners ATG5 and ATG12 are required for plasma membrane repair through a pathway independent of macroautophagy. ATG16L1 is required for lysosome fusion with the plasma membrane and blebbing responses that promote membrane repair. ATG16L1 deficiency causes accumulation of cholesterol in lysosomes that contributes to defective membrane repair. Cell-to-cell spread by Listeria monocytogenes requires membrane damage by the bacterial toxin listeriolysin O, which is restricted by ATG16L1-dependent membrane repair. Cells harbouring the ATG16L1 T300A allele associated with inflammatory bowel disease were also found to accumulate cholesterol and be defective in repair, linking a common inflammatory disease to plasma membrane integrity. Thus, plasma membrane repair could be an important therapeutic target for the treatment of bacterial infections and inflammatory disorders.

PMID: 30478389 [PubMed - in process]



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Type VI secretion system baseplate.

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Type VI secretion system baseplate.

Nat Microbiol. 2018 12;3(12):1330-1331

Authors: Davidson AR, Maxwell KL

Abstract

PMID: 30478386 [PubMed - indexed for MEDLINE]



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Dual embryonic origin of the mammalian enteric nervous system.

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Dual embryonic origin of the mammalian enteric nervous system.

Dev Biol. 2018 Nov 22;:

Authors: Brokhman I, Xu J, Coles BLK, Razavi R, Engert S, Lickert H, Babona-Pilipos R, Morshead CM, Sibley E, Chen C, van der Kooy D

Abstract
The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.

PMID: 30472119 [PubMed - as supplied by publisher]



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Human Oligodendrogenic Neural Progenitor Cells Delivered with Chondroitinase ABC Facilitate Functional Repair of Chronic Spinal Cord Injury.

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Human Oligodendrogenic Neural Progenitor Cells Delivered with Chondroitinase ABC Facilitate Functional Repair of Chronic Spinal Cord Injury.

Stem Cell Reports. 2018 Nov 09;:

Authors: Nori S, Khazaei M, Ahuja CS, Yokota K, Ahlfors JE, Liu Y, Wang J, Shibata S, Chio J, Hettiaratchi MH, Führmann T, Shoichet MS, Fehlings MG

Abstract
Treatment of chronic spinal cord injury (SCI) is challenging due to cell loss, cyst formation, and the glial scar. Previously, we reported on the therapeutic potential of a neural progenitor cell (NPC) and chondroitinase ABC (ChABC) combinatorial therapy for chronic SCI. However, the source of NPCs and delivery system required for ChABC remained barriers to clinical application. Here, we investigated directly reprogrammed human NPCs biased toward an oligodendrogenic fate (oNPCs) in combination with sustained delivery of ChABC using an innovative affinity release strategy in a crosslinked methylcellulose biomaterial for the treatment of chronic SCI in an immunodeficient rat model. This combinatorial therapy increased long-term survival of oNPCs around the lesion epicenter, facilitated greater oligodendrocyte differentiation, remyelination of the spared axons by engrafted oNPCs, enhanced synaptic connectivity with anterior horn cells and neurobehavioral recovery. This combinatorial therapy is a promising strategy to regenerate the chronically injured spinal cord.

PMID: 30472009 [PubMed - as supplied by publisher]



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Allosteric Modulation of Binding Specificity by Alternative Packing of Protein Cores.

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Allosteric Modulation of Binding Specificity by Alternative Packing of Protein Cores.

J Mol Biol. 2018 Nov 21;:

Authors: Ben-David M, Huang H, Sun MGF, Corbi-Verge C, Petsalaki E, Liu K, Gfeller D, Garg P, Tempel W, Sochirca I, Shifman JM, Davidson A, Min J, Kim PM, Sidhu SS

Abstract
Hydrophobic cores are often viewed as tightly packed and rigid, but they do show some plasticity and could thus be attractive targets for protein design. Here we explored the role of different functional pressures on the core packing and ligand recognition of the SH3 domain from human Fyn tyrosine kinase. We randomized the hydrophobic core and used phage display to select variants that bound to each of three distinct ligands. The three evolved groups showed remarkable differences in core composition, illustrating the effect of different selective pressures on the core. Changes in the core did not significantly alter protein stability, but were linked closely to changes in binding affinity and specificity. Structural analysis and molecular dynamics simulations revealed the structural basis for altered specificity. The evolved domains had significantly reduced core volumes, which in turn induced increased backbone flexibility. These motions were propagated from the core to the binding surface and induced significant conformational changes. These results show that alternative core packing and consequent allosteric modulation of binding interfaces could be used to engineer proteins with novel functions.

PMID: 30471255 [PubMed - as supplied by publisher]



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Initial cell maturity changes following transplantation in a hyaluronan-based hydrogel and impacts therapeutic success in the stroke-injured rodent brain.

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Initial cell maturity changes following transplantation in a hyaluronan-based hydrogel and impacts therapeutic success in the stroke-injured rodent brain.

Biomaterials. 2018 Nov 15;192:309-322

Authors: Payne SL, Tuladhar A, Obermeyer JM, Varga BV, Teal CJ, Morshead CM, Nagy A, Shoichet MS

Abstract
Ischemic stroke results in a loss of neurons for which there are no available clinical strategies to stimulate regeneration. While preclinical studies have demonstrated that functional recovery can be obtained by transplanting an exogenous source of neural progenitors into the brain, it remains unknown at which stage of neuronal maturity cells will provide the most benefit. We investigated the role of neuronal maturity on cell survival, differentiation, and long-term sensorimotor recovery in stroke-injured rats using a population of human cortically-specified neuroepithelial progenitor cells (cNEPs) delivered in a biocompatible, bioresorbable hyaluronan/methylcellulose hydrogel. We demonstrate that transplantation of immature cNEPs result in the greatest tissue and functional repair, relative to transplantation of more mature neurons. The transplantation process itself resulted in the least cell death and phenotypic changes in the immature cNEPs, and the greatest acute cell death in the mature cells. The latter negatively impacted host tissue and negated any potential positive effects associated with cell maturity and the hydrogel vehicle, which itself showed some functional and tissue benefit. Moreover, we show that more mature cell populations are drastically altered during the transplantation process itself. The phenotype of the cells before and after transplantation had an enormous impact on their survival and the consequent tissue and behavioral response, emphasizing the importance of characterizing injected cells in transplantation studies more broadly.

PMID: 30468998 [PubMed - as supplied by publisher]



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Protocadherin-1 is essential for cell entry by New World hantaviruses.

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Protocadherin-1 is essential for cell entry by New World hantaviruses.

Nature. 2018 Nov;563(7732):559-563

Authors: Jangra RK, Herbert AS, Li R, Jae LT, Kleinfelter LM, Slough MM, Barker SL, Guardado-Calvo P, Román-Sosa G, Dieterle ME, Kuehne AI, Muena NA, Wirchnianski AS, Nyakatura EK, Fels JM, Ng M, Mittler E, Pan J, Bharrhan S, Wec AZ, Lai JR, Sidhu SS, Tischler ND, Rey FA, Moffat J, Brummelkamp TR, Wang Z, Dye JM, Chandran K

Abstract
The zoonotic transmission of hantaviruses from their rodent hosts to humans in North and South America is associated with a severe and frequently fatal respiratory disease, hantavirus pulmonary syndrome (HPS)1,2. No specific antiviral treatments for HPS are available, and no molecular determinants of in vivo susceptibility to hantavirus infection and HPS are known. Here we identify the human asthma-associated gene protocadherin-1 (PCDH1)3-6 as an essential determinant of entry and infection in pulmonary endothelial cells by two hantaviruses that cause HPS, Andes virus (ANDV) and Sin Nombre virus (SNV). In vitro, we show that the surface glycoproteins of ANDV and SNV directly recognize the outermost extracellular repeat domain of PCDH1-a member of the cadherin superfamily7,8-to exploit PCDH1 for entry. In vivo, genetic ablation of PCDH1 renders Syrian golden hamsters highly resistant to a usually lethal ANDV challenge. Targeting PCDH1 could provide strategies to reduce infection and disease caused by New World hantaviruses.

PMID: 30464266 [PubMed - in process]



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