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Recent Publications

Preclinical evaluation of taxane-binding peptide-modified polymeric micelles loaded with docetaxel in an orthotopic breast cancer mouse model.

Preclinical evaluation of taxane-binding peptide-modified polymeric micelles loaded with docetaxel in an orthotopic breast cancer mouse model.

Biomaterials. 2017 Jan 26;123:39-47

Authors: Logie J, Ganesh AN, Aman AM, Al-Awar RS, Shoichet MS

Abstract
We developed a novel taxane-binding peptide (TBP) modified, biodegradable polymeric micelle that overcomes limitations of drug loading and poor serum stability typically seen with particle delivery, leading to enhanced pharmacokinetics and tumor distribution of docetaxel (DTX). The use of the taxane-binding peptide to increase docetaxel loading is particularly compelling as it takes advantage of a known intracellular binding mechanism in a new way. Docetaxel is a potent chemotherapeutic with a therapeutic index often limited by the toxicity of the excipients that are necessary to enhance its solubility for intravenous delivery. Our polymeric micelle has terminal furan groups that enable facile antibody Fab conjugation by Diels-Alder chemistry for targeted delivery. Compared to the conventional ethanolic polysorbate 80 formulation (Free DTX), our nanoparticle (NP DTX) formulation exhibited a two-fold increase in exposure and tumor accumulation. Notably, the reduced toxicity of the NP DTX formulation increased the therapeutic index and allowed for higher dosing regimens, with a maximum tolerated dose (MTD) 1.6-fold higher than that of the Free DTX formulation, which is significant and similar to enhancements observed in clinical products for docetaxel and other drugs. These improved properties led to enhanced mouse survival in an orthotopic model of breast cancer; however, the targeted formulation of Fab-NP DTX did not further improve efficacy. Together, these results clearly demonstrate the benefits of the TBP-modified polymeric micelles as promising carriers for docetaxel.

PMID: 28161682 [PubMed - as supplied by publisher]



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Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

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Multilayered Control of Alternative Splicing Regulatory Networks by Transcription Factors.

Mol Cell. 2017 Feb 02;65(3):539-553.e7

Authors: Han H, Braunschweig U, Gonatopoulos-Pournatzis T, Weatheritt RJ, Hirsch CL, Ha KC, Radovani E, Nabeel-Shah S, Sterne-Weiler T, Wang J, O'Hanlon D, Pan Q, Ray D, Zheng H, Vizeacoumar F, Datti A, Magomedova L, Cummins CL, Hughes TR, Greenblatt JF, Wrana JL, Moffat J, Blencowe BJ

Abstract
Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large "missing cache" of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

PMID: 28157508 [PubMed - in process]



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Genome-Scale Networks Link Neurodegenerative Disease Genes to α-Synuclein through Specific Molecular Pathways.

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Genome-Scale Networks Link Neurodegenerative Disease Genes to α-Synuclein through Specific Molecular Pathways.

Cell Syst. 2017 Jan 25;:

Authors: Khurana V, Peng J, Chung CY, Auluck PK, Fanning S, Tardiff DF, Bartels T, Koeva M, Eichhorn SW, Benyamini H, Lou Y, Nutter-Upham A, Baru V, Freyzon Y, Tuncbag N, Costanzo M, San Luis BJ, Schöndorf DC, Barrasa MI, Ehsani S, Sanjana N, Zhong Q, Gasser T, Bartel DP, Vidal M, Deleidi M, Boone C, Fraenkel E, Berger B, Lindquist S

Abstract
Numerous genes and molecular pathways are implicated in neurodegenerative proteinopathies, but their inter-relationships are poorly understood. We systematically mapped molecular pathways underlying the toxicity of alpha-synuclein (α-syn), a protein central to Parkinson's disease. Genome-wide screens in yeast identified 332 genes that impact α-syn toxicity. To "humanize" this molecular network, we developed a computational method, TransposeNet. This integrates a Steiner prize-collecting approach with homology assignment through sequence, structure, and interaction topology. TransposeNet linked α-syn to multiple parkinsonism genes and druggable targets through perturbed protein trafficking and ER quality control as well as mRNA metabolism and translation. A calcium signaling hub linked these processes to perturbed mitochondrial quality control and function, metal ion transport, transcriptional regulation, and signal transduction. Parkinsonism gene interaction profiles spatially opposed in the network (ATP13A2/PARK9 and VPS35/PARK17) were highly distinct, and network relationships for specific genes (LRRK2/PARK8, ATXN2, and EIF4G1/PARK18) were confirmed in patient induced pluripotent stem cell (iPSC)-derived neurons. This cross-species platform connected diverse neurodegenerative genes to proteinopathy through specific mechanisms and may facilitate patient stratification for targeted therapy.

PMID: 28131822 [PubMed - as supplied by publisher]



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Functional Analysis of Kinases and Transcription Factors in Saccharomyces cerevisiae Using an Integrated Overexpression Library.

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Functional Analysis of Kinases and Transcription Factors in Saccharomyces cerevisiae Using an Integrated Overexpression Library.

G3 (Bethesda). 2017 Jan 25;:

Authors: Youn JY, Friesen H, Nguyen Ba AN, Liang W, Messier V, Cox MJ, Moses AM, Andrews B

Abstract
Kinases and transcription factors (TFs) are key modulators of important signaling pathways and their activities underlie the proper function of many basic cellular processes such as cell division, differentiation and development. Changes in kinase and TF dosage are often associated with disease, yet a systematic assessment of the cellular phenotypes caused by the combined perturbation of kinases and TFs has not been undertaken. We used a reverse-genetics approach to study the phenotypic consequences of kinase and TF overexpression in the budding yeast, Saccharomyces cerevisiae We constructed a collection of strains expressing stably integrated inducible alleles of kinases and TFs and used a variety of assays to characterize the phenotypes caused by TF and kinase overexpression. We used the Synthetic Genetic Array (SGA) method to examine dosage-dependent genetic interactions (GIs) between 239 gain-of-function (overexpression) alleles of TFs and six loss-of-function and seven over-expression kinase alleles, the former identifying Synthetic Dosage Lethal (SDL) interactions and the latter testing a genetic interaction we call Double Dosage Lethality (DDL). We identified and confirmed 94 genetic interactions between 65 overexpression alleles of TFs and nine kinase alleles. Follow-up experiments validated regulatory relationships between genetically interacting pairs (Cdc28 - Stb1 and Pho85 - Pdr1), suggesting that genetic interaction studies involving overexpression alleles of regulatory proteins will be a rich source of new functional information.

PMID: 28122947 [PubMed - as supplied by publisher]



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Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth.

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Drosophila larvae synthesize the putative oncometabolite L-2-hydroxyglutarate during normal developmental growth.

Proc Natl Acad Sci U S A. 2017 Jan 23;:

Authors: Li H, Chawla G, Hurlburt AJ, Sterrett MC, Zaslaver O, Cox J, Karty JA, Rosebrock AP, Caudy AA, Tennessen JM

Abstract
L-2-hydroxyglutarate (L-2HG) has emerged as a putative oncometabolite that is capable of inhibiting enzymes involved in metabolism, chromatin modification, and cell differentiation. However, despite the ability of L-2HG to interfere with a broad range of cellular processes, this molecule is often characterized as a metabolic waste product. Here, we demonstrate that Drosophila larvae use the metabolic conditions established by aerobic glycolysis to both synthesize and accumulate high concentrations of L-2HG during normal developmental growth. A majority of the larval L-2HG pool is derived from glucose and dependent on the Drosophila estrogen-related receptor (dERR), which promotes L-2HG synthesis by up-regulating expression of the Drosophila homolog of lactate dehydrogenase (dLdh). We also show that dLDH is both necessary and sufficient for directly synthesizing L-2HG and the Drosophila homolog of L-2-hydroxyglutarate dehydrogenase (dL2HGDH), which encodes the enzyme that breaks down L-2HG, is required for stage-specific degradation of the L-2HG pool. In addition, dLDH also indirectly promotes L-2HG accumulation via synthesis of lactate, which activates a metabolic feed-forward mechanism that inhibits dL2HGDH activity and stabilizes L-2HG levels. Finally, we use a genetic approach to demonstrate that dLDH and L-2HG influence position effect variegation and DNA methylation, suggesting that this compound serves to coordinate glycolytic flux with epigenetic modifications. Overall, our studies demonstrate that growing animal tissues synthesize L-2HG in a controlled manner, reveal a mechanism that coordinates glucose catabolism with L-2HG synthesis, and establish the fly as a unique model system for studying the endogenous functions of L-2HG during cell growth and proliferation.

PMID: 28115720 [PubMed - as supplied by publisher]



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High Density or Urban Sprawl: What Works Best in Biology?

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High Density or Urban Sprawl: What Works Best in Biology?

ACS Nano. 2017 Jan 23;:

Authors: Oreopoulos J, Gray-Owen SD, Yip CM

Abstract
With new approaches in imaging-from new tools or reagents to processing algorithms-come unique opportunities and challenges to our understanding of biological processes, structures, and dynamics. Although innovations in super-resolution imaging are affording novel perspectives into how molecules structurally associate and localize in response to, or in order to initiate, specific signaling events in the cell, questions arise as to how to interpret these observations in the context of biological function. Just as each neighborhood in a city has its own unique vibe, culture, and indeed density, recent work has shown that membrane receptor behavior and action is governed by their localization and association state. There is tremendous potential in developing strategies for tracking how the populations of these molecular neighborhoods change dynamically.

PMID: 28112892 [PubMed - as supplied by publisher]



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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 Jan 20;:

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 - as supplied by publisher]



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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 Dec 15;30(24):2696-2709

Authors: Kwan J, Sczaniecka A, Arash EH, 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 - in process]



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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 - in process]



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