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VAPs and ACBD5 tether peroxisomes to the ER for peroxisome maintenance and lipid homeostasis.

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VAPs and ACBD5 tether peroxisomes to the ER for peroxisome maintenance and lipid homeostasis.

J Cell Biol. 2017 Feb;216(2):367-377

Authors: Hua R, Cheng D, Coyaud É, Freeman S, Di Pietro E, Wang Y, Vissa A, Yip CM, Fairn GD, Braverman N, Brumell JH, Trimble WS, Raught B, Kim PK

Abstract
Lipid exchange between the endoplasmic reticulum (ER) and peroxisomes is necessary for the synthesis and catabolism of lipids, the trafficking of cholesterol, and peroxisome biogenesis in mammalian cells. However, how lipids are exchanged between these two organelles is not understood. In this study, we report that the ER-resident VAMP-associated proteins A and B (VAPA and VAPB) interact with the peroxisomal membrane protein acyl-CoA binding domain containing 5 (ACBD5) and that this interaction is required to tether the two organelles together, thereby facilitating the lipid exchange between them. Depletion of either ACBD5 or VAP expression results in increased peroxisome mobility, suggesting that VAP-ACBD5 complex acts as the primary ER-peroxisome tether. We also demonstrate that tethering of peroxisomes to the ER is necessary for peroxisome growth, the synthesis of plasmalogen phospholipids, and the maintenance of cellular cholesterol levels. Collectively, our data highlight the importance of VAP-ACBD5-mediated contact between the ER and peroxisomes for organelle maintenance and lipid homeostasis.

PMID: 28108526 [PubMed - indexed for MEDLINE]



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Creation of Phosphotyrosine Superbinders by Directed Evolution of an SH2 Domain.

Creation of Phosphotyrosine Superbinders by Directed Evolution of an SH2 Domain.

Methods Mol Biol. 2017;1555:225-254

Authors: Huang H, Kaneko T, Sidhu SS, Li SS

Abstract
Commercial antibodies raised against phosphotyrosine have been widely used as reagents to detect or isolate tyrosine-phosphorylated proteins from cellular samples. However, these antibodies are costly and are not amenable to in-house production in an academic lab setting. In this chapter, we describe a method to generate super-high affinity SH2 domains, dubbed the phosphotyrosine superbinders, by evolving a natural SH2 domain using the phage display technology. The superbinders are stable and can be easily produced in Escherichia coli in large quantities. The strategy presented here may also be applied to other protein domains to generate domain variants with markedly enhanced affinities for a specific post-translational modification.

PMID: 28092036 [PubMed - in process]



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DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2.

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DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2.

Genes Dev. 2016 12 15;30(24):2696-2709

Authors: Kwan J, Sczaniecka A, Heidary Arash E, Nguyen L, Chen CC, Ratkovic S, Klezovitch O, Attisano L, McNeill H, Emili A, Vasioukhin V

Abstract
Disruption of apical-basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5(-/-) tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms.

PMID: 28087714 [PubMed - indexed for MEDLINE]



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Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress.

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Chaperone-Mediated Autophagy Protein BAG3 Negatively Regulates Ebola and Marburg VP40-Mediated Egress.

PLoS Pathog. 2017 Jan;13(1):e1006132

Authors: Liang J, Sagum CA, Bedford MT, Sidhu SS, Sudol M, Han Z, Harty RN

Abstract
Ebola (EBOV) and Marburg (MARV) viruses are members of the Filoviridae family which cause outbreaks of hemorrhagic fever. The filovirus VP40 matrix protein is essential for virus assembly and budding, and its PPxY L-domain motif interacts with WW-domains of specific host proteins, such as Nedd4 and ITCH, to facilitate the late stage of virus-cell separation. To identify additional WW-domain-bearing host proteins that interact with VP40, we used an EBOV PPxY-containing peptide to screen an array of 115 mammalian WW-domain-bearing proteins. Using this unbiased approach, we identified BCL2 Associated Athanogene 3 (BAG3), a member of the BAG family of molecular chaperone proteins, as a specific VP40 PPxY interactor. Here, we demonstrate that the WW-domain of BAG3 interacts with the PPxY motif of both EBOV and MARV VP40 and, unexpectedly, inhibits budding of both eVP40 and mVP40 virus-like particles (VLPs), as well as infectious VSV-EBOV recombinants. BAG3 is a stress induced protein that regulates cellular protein homeostasis and cell survival through chaperone-mediated autophagy (CMA). Interestingly, our results show that BAG3 alters the intracellular localization of VP40 by sequestering VP40 away from the plasma membrane. As BAG3 is the first WW-domain interactor identified that negatively regulates budding of VP40 VLPs and infectious virus, we propose that the chaperone-mediated autophagy function of BAG3 represents a specific host defense strategy to counteract the function of VP40 in promoting efficient egress and spread of virus particles.

PMID: 28076420 [PubMed - indexed for MEDLINE]



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Using the Electronic Medical Record to Identify Patients at High Risk for Frequent Emergency Department Visits and High System Costs.

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Using the Electronic Medical Record to Identify Patients at High Risk for Frequent Emergency Department Visits and High System Costs.

Am J Med. 2017 May;130(5):601.e17-601.e22

Authors: Frost DW, Vembu S, Wang J, Tu K, Morris Q, Abrams HB

Abstract
BACKGROUND: A small proportion of patients account for a high proportion of healthcare use. Accurate preemptive identification may facilitate tailored intervention. We sought to determine whether machine learning techniques using text from a family practice electronic medical record can be used to predict future high emergency department use and total costs by patients who are not yet high emergency department users or high cost to the healthcare system.
METHODS: Text from fields of the cumulative patient profile within an electronic medical record of 43,111 patients was indexed. Separate training and validation cohorts were created. After processing, 11,905 words were used to fit a logistic regression model. The primary outcomes of interest in the 12 months after prediction were 3 or more emergency department visits and being in the top 5% in healthcare expenditures. Outcomes were assessed through linkage to administrative databases housed at the Institute for Clinical Evaluative Sciences.
RESULTS: In the model to predict frequent emergency department visits, after excluding patients who were high emergency department users in the previous year, the area under the receiver operating characteristic curve was 0.71. By using the same methodology, the model to predict the top 5% in total system costs had an area under the receiver operating characteristic curve of 0.76.
CONCLUSIONS: Machine learning techniques can be applied to analyze free text contained in electronic medical records. This dataset is more predictive of patients who will generate future high costs than future emergency department visits. It remains to be seen whether these predictions can be used to reduce costs by early interventions in this cohort of patients.

PMID: 28065773 [PubMed - indexed for MEDLINE]



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A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome.

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A Global Analysis of the Receptor Tyrosine Kinase-Protein Phosphatase Interactome.

Mol Cell. 2016 Dec 21;:

Authors: Yao Z, Darowski K, St-Denis N, Wong V, Offensperger F, Villedieu A, Amin S, Malty R, Aoki H, Guo H, Xu Y, Iorio C, Kotlyar M, Emili A, Jurisica I, Neel BG, Babu M, Gingras AC, Stagljar I

Abstract
Receptor tyrosine kinases (RTKs) and protein phosphatases comprise protein families that play crucial roles in cell signaling. We used two protein-protein interaction (PPI) approaches, the membrane yeast two-hybrid (MYTH) and the mammalian membrane two-hybrid (MaMTH), to map the PPIs between human RTKs and phosphatases. The resulting RTK-phosphatase interactome reveals a considerable number of previously unidentified interactions and suggests specific roles for different phosphatase families. Additionally, the differential PPIs of some protein tyrosine phosphatases (PTPs) and their mutants suggest diverse mechanisms of these PTPs in the regulation of RTK signaling. We further found that PTPRH and PTPRB directly dephosphorylate EGFR and repress its downstream signaling. By contrast, PTPRA plays a dual role in EGFR signaling: besides facilitating EGFR dephosphorylation, it enhances downstream ERK signaling by activating SRC. This comprehensive RTK-phosphatase interactome study provides a broad and deep view of RTK signaling.

PMID: 28065597 [PubMed - as supplied by publisher]



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Achieving Efficient Manufacturing and Quality Assurance through Synthetic Cell Therapy Design.

Achieving Efficient Manufacturing and Quality Assurance through Synthetic Cell Therapy Design.

Cell Stem Cell. 2017 Jan 05;20(1):13-17

Authors: Lipsitz YY, Bedford P, Davies AH, Timmins NE, Zandstra PW

Abstract
New methods to manipulate gene and cell state can be used to engineer cell functionality, simplify quality assessment, and enhance manufacturability. These strategies could help overcome unresolved cell therapy manufacturing challenges and complement frameworks to design quality into these complex cellular systems, ultimately increasing patient access to living therapeutics.

PMID: 28061350 [PubMed - in process]



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Guiding principles for a successful multidisciplinary research collaboration.

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Guiding principles for a successful multidisciplinary research collaboration.

Future Sci OA. 2015 Nov;1(3):FSO7

Authors: Lustig LC, Ponzielli R, Tang PS, Sathiamoorthy S, Inamoto I, Shin JA, Penn LZ, Chan WC

PMID: 28031882 [PubMed]



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Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins.

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Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins.

EMBO J. 2017 Feb 15;36(4):549-564

Authors: Stolz A, Putyrski M, Kutle I, Huber J, Wang C, Major V, Sidhu SS, Youle RJ, Rogov VV, Dötsch V, Ernst A, Dikic I

Abstract
Autophagy is a cellular surveillance pathway that balances metabolic and energy resources and transports specific cargos, including damaged mitochondria, other broken organelles, or pathogens for degradation to the lysosome. Central components of autophagosomal biogenesis are six members of the LC3 and GABARAP family of ubiquitin-like proteins (mATG8s). We used phage display to isolate peptides that possess bona fide LIR (LC3-interacting region) properties and are selective for individual mATG8 isoforms. Sensitivity of the developed sensors was optimized by multiplication, charge distribution, and fusion with a membrane recruitment (FYVE) or an oligomerization (PB1) domain. We demonstrate the use of the engineered peptides as intracellular sensors that recognize specifically GABARAP, GABL1, GABL2, and LC3C, as well as a bispecific sensor for LC3A and LC3B. By using an LC3C-specific sensor, we were able to monitor recruitment of endogenous LC3C to Salmonella during xenophagy, as well as to mitochondria during mitophagy. The sensors are general tools to monitor the fate of mATG8s and will be valuable in decoding the biological functions of the individual LC3/GABARAPs.

PMID: 28028054 [PubMed - indexed for MEDLINE]



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Stereotypical Escape Behavior in Caenorhabditis elegans Allows Quantification of Effective Heat Stimulus Level.

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Stereotypical Escape Behavior in Caenorhabditis elegans Allows Quantification of Effective Heat Stimulus Level.

PLoS Comput Biol. 2016 Dec;12(12):e1005262

Authors: Leung K, Mohammadi A, Ryu WS, Nemenman I

Abstract
A goal of many sensorimotor studies is to quantify the stimulus-behavioral response relation for specific organisms and specific sensory stimuli. This is especially important to do in the context of painful stimuli since most animals in these studies cannot easily communicate to us their perceived levels of such noxious stimuli. Thus progress on studies of nociception and pain-like responses in animal models depends crucially on our ability to quantitatively and objectively infer the sensed levels of these stimuli from animal behaviors. Here we develop a quantitative model to infer the perceived level of heat stimulus from the stereotyped escape response of individual nematodes Caenorhabditis elegans stimulated by an IR laser. The model provides a method for quantification of analgesic-like effects of chemical stimuli or genetic mutations in C. elegans. We test ibuprofen-treated worms and a TRPV (transient receptor potential) mutant, and we show that the perception of heat stimuli for the ibuprofen treated worms is lower than the wild-type. At the same time, our model shows that the mutant changes the worm's behavior beyond affecting the thermal sensory system. Finally, we determine the stimulus level that best distinguishes the analgesic-like effects and the minimum number of worms that allow for a statistically significant identification of these effects.

PMID: 28027302 [PubMed - indexed for MEDLINE]



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