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Biosynthetic Oligoclonal Antivenom (BOA) for Snakebite and Next-Generation Treatments for Snakebite Victims.

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Biosynthetic Oligoclonal Antivenom (BOA) for Snakebite and Next-Generation Treatments for Snakebite Victims.

Toxins (Basel). 2018 Dec 13;10(12):

Authors: Kini RM, Sidhu SS, Laustsen AH

Abstract
Snakebite envenoming is a neglected tropical disease that each year claims the lives of 80,000⁻140,000 victims worldwide. The only effective treatment against envenoming involves intravenous administration of antivenoms that comprise antibodies that have been isolated from the plasma of immunized animals, typically horses. The drawbacks of such conventional horse-derived antivenoms include their propensity for causing allergenic adverse reactions due to their heterologous and foreign nature, an inability to effectively neutralize toxins in distal tissue, a low content of toxin-neutralizing antibodies, and a complex manufacturing process that is dependent on husbandry and procurement of snake venoms. In recent years, an opportunity to develop a fundamentally novel type of antivenom has presented itself. By using modern antibody discovery strategies, such as phage display selection, and repurposing small molecule enzyme inhibitors, next-generation antivenoms that obviate the drawbacks of existing plasma-derived antivenoms could be developed. This article describes the conceptualization of a novel therapeutic development strategy for biosynthetic oligoclonal antivenom (BOA) for snakebites based on recombinantly expressed oligoclonal mixtures of human monoclonal antibodies, possibly combined with repurposed small molecule enzyme inhibitors.

PMID: 30551565 [PubMed - in process]



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Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response.

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Functional genomic characterization of a synthetic anti-HER3 antibody reveals a role for ubiquitination by RNF41 in the anti-proliferative response.

J Biol Chem. 2018 Dec 06;:

Authors: Turowec JP, Lau EWT, Wang X, Brown KR, Fellouse FA, Jawanda KK, Pan J, Moffat J, Sidhu S

Abstract
Dysregulation of the ErbB family of receptor tyrosine kinases is involved in the progression of many cancers. Antibodies targeting the dimerization domains of family members EGFR and HER2 are approve cancer therapeutics, but efficacy is restricted to a subset of tumors and resistance often develops in response to treatment. A third family member, HER3, heterodimerizes with both EGFR and HER2 and has also been implicated in cancer. Consequently, there is strong interest in developing antibodies that target HER3, but to date, no therapeutics have been approved. To aid the development of anti-HER3 antibodies as cancer therapeutics, we combined antibody engineering and functional genomics screens to identify putative mechanisms of resistance or synthetic lethality with antibody-mediated anti-proliferative effects. We developed a synthetic antibody called IgG 95, which binds to HER3 and promotes ubiquitination, internalization and receptor down-regulation. Using an shRNA library targeting enzymes in the ubiquitin proteasome system, we screened for genes that effect response to IgG 95 and uncovered the E3 ubiquitin ligase RNF41 as a driver of IgG 95 anti-proliferative activity. RNF41 has been shown previously to regulate HER3 levels under normal conditions and we now show that it is also responsible for down-regulation of HER3 upon treatment with IgG 95. Moreover, our findings suggest that down-regulation of RNF41 itself may be a mechanism for acquired resistance to treatment with IgG 95 and perhaps other anti-HER3 antibodies. Our work deepens our understanding of HER3 signaling by uncovering the mechanistic basis for the anti-proliferative effects of potential anti-HER3 antibody therapeutics.

PMID: 30523157 [PubMed - as supplied by publisher]



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Rapid and Accurate Structure-Based Therapeutic Peptide Design using GPU Accelerated Thermodynamic Integration.

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Rapid and Accurate Structure-Based Therapeutic Peptide Design using GPU Accelerated Thermodynamic Integration.

Proteins. 2018 Dec 06;:

Authors: Garton M, Corbi-Verge C, Hu Y, Nim S, Tarasova N, Sherborne B, Kim PM

Abstract
Peptide-based therapeutics are an alternative to small molecule drugs as they offer superior specificity, lower toxicity and easy synthesis. Here we present an approach that leverages the dramatic performance increase afforded by the recent arrival of GPU accelerated thermodynamic integration (TI). GPU TI facilitates very fast, highly accurate binding affinity optimization of peptides against therapeutic targets. We benchmarked TI predictions using published peptide binding optimization studies. Prediction of mutations involving charged side-chains was found to be less accurate than for non-charged, and use of a more complex 3-step TI protocol was found to boost accuracy in these cases. Using the 3-step protocol for non-charged side-chains either had no effect or was detrimental. We use the benchmarked pipeline to optimize a peptide binding to our recently discovered cancer target: EME1. TI calculations predict beneficial mutations using both canonical and non-canonical amino acids. We validate these predictions using fluorescence polarization and confirm that binding affinity is increased. We further demonstrate that this increase translates to a significant reduction in pancreatic cancer cell viability. This article is protected by copyright. All rights reserved.

PMID: 30520126 [PubMed - as supplied by publisher]



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A chemical defence against phage infection.

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A chemical defence against phage infection.

Nature. 2018 Dec 05;:

Authors: Kronheim S, Daniel-Ivad M, Duan Z, Hwang S, Wong AI, Mantel I, Nodwell JR, Maxwell KL

Abstract
The arms race between bacteria and the phages that infect them drives the continual evolution of diverse anti-phage defences. Previously described anti-phage systems have highly varied defence mechanisms1-11; however, all mechanisms rely on protein components to mediate defence. Here we report a chemical anti-phage defence system that is widespread in Streptomyces. We show that three naturally produced molecules that insert into DNA are able to block phage replication, whereas molecules that target DNA by other mechanisms do not. Because double-stranded DNA phages are the most numerous group in the biosphere and the production of secondary metabolites by bacteria is ubiquitous12, this mechanism of anti-phage defence probably has a major evolutionary role in shaping bacterial communities.

PMID: 30518855 [PubMed - as supplied by publisher]



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Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.

Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins.

MBio. 2018 Dec 04;9(6):

Authors: Lee J, Mir A, Edraki A, Garcia B, Amrani N, Lou HE, Gainetdinov I, Pawluk A, Ibraheim R, Gao XD, Liu P, Davidson AR, Maxwell KL, Sontheimer EJ

Abstract
In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017). We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8-14, 2017, https://doi.org/10.1016/j.molcel.2017.09.002; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12-17, 2018, https://doi.org/10.1038/nrmicro.2017.120; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120-127, 2017, https://doi.org/10.1016/j.mib.2017.06.003). We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829-1838.e9, 2016, https://doi.org/10.1016/j.cell.2016.11.017) against Neisseria meningitidis Cas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains of Haemophilus parainfluenzae and Simonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357-365, 2018, https://doi.org/10.1021/acschembio.7b00855). We characterize the type II-C Cas9 orthologs from H. parainfluenzae and S. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. The S. muelleri Acr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs from H. parainfluenzae and S. muelleri reveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.IMPORTANCE As one of their countermeasures against CRISPR-Cas immunity, bacteriophages have evolved natural inhibitors known as anti-CRISPR (Acr) proteins. Despite the existence of such examples for type II CRISPR-Cas systems, we currently know relatively little about the breadth of Cas9 inhibitors, and most of their direct Cas9 targets are uncharacterized. In this work we identify two new type II-C anti-CRISPRs and their cognate Cas9 orthologs, validate their functionality in vitro and in bacteria, define their inhibitory spectrum against a panel of Cas9 orthologs, demonstrate that they act before Cas9 DNA binding, and document their utility as off-switches for Cas9-based tools in mammalian applications. The discovery of diverse anti-CRISPRs, the mechanistic analysis of their cognate Cas9s, and the definition of Acr inhibitory mechanisms afford deeper insight into the interplay between Cas9 orthologs and their inhibitors and provide greater scope for exploiting Acrs for CRISPR-based genome engineering.

PMID: 30514786 [PubMed - in process]



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Mapping a diversity of genetic interactions in yeast.

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Mapping a diversity of genetic interactions in yeast.

Curr Opin Syst Biol. 2017 Dec;6:14-21

Authors: van Leeuwen J, Boone C, Andrews BJ

Abstract
Genetic interactions occur when the combination of multiple mutations yields an unexpected phenotype, and they may confound our ability to fully understand the genetic mechanisms underlying complex diseases. Genetic interactions are challenging to study because there are millions of possible different variant combinations within a given genome. Consequently, they have primarily been systematically explored in unicellular model organisms, such as yeast, with a focus on pairwise genetic interactions between loss-of-function alleles. However, there are many different types of genetic interactions, such as those occurring between gain-of-function or heterozygous mutations. Here, we review recent advances made in the systematic analysis of such diverse genetic interactions in yeast, and briefly discuss how similar studies could be undertaken in human cells.

PMID: 30505984 [PubMed]



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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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