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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. 2016 Dec 27;:

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 - as supplied by publisher]



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

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 - in process]



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Parkinson's disease-associated GPR37 receptor regulates cocaine-mediated synaptic depression in corticostriatal synapses.

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Parkinson's disease-associated GPR37 receptor regulates cocaine-mediated synaptic depression in corticostriatal synapses.

Neurosci Lett. 2016 Dec 19;:

Authors: Rial D, Morató X, Real JI, Gonçalves FQ, Stagljar I, Pereira FC, Fernández-Dueñas V, Cunha RA, Ciruela F

Abstract
GPR37 is an orphan G protein-coupled receptor highly expressed in the brain. The precise function of GPR37 is still unknown, but a number of evidences indicate it modulates the dopaminergic system. Here, we aimed to determine the role of GPR37 on the control of cocaine-mediated electrophysiological effects (synaptic transmission and short-term plasticity) in corticostriatal synapses. Accordingly, we evaluated basal synaptic transmission and paired-pulse stimulation (PPS) in wild-type and GPR37KO mice slices. Regardless of the genotype, a low concentration of cocaine (2μM) did not modify basal synaptic transmission. Conversely, a higher dose of cocaine (30μM) decreased synaptic transmission in both genotypes, although with different intensities: approximately 30% in slices from wild-type mice and 45% in slices from GPR37-KO mice. On the other hand, no differences in PPS ratio were observed between wild-type and GPR37-KO cocaine-treated mice. Overall, our data suggest that GPR37 is involved in cocaine-induced modification of basal synaptic transmission without modifying cocaine effects in short-term plasticity.

PMID: 28007645 [PubMed - as supplied by publisher]



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Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome.

Discovery of short linear motif-mediated interactions through phage display of intrinsically disordered regions of the human proteome.

FEBS J. 2016 Dec 21;:

Authors: Davey NE, Seo MH, Yadav VK, Jeon J, Nim S, Krystkowiak I, Blikstad C, Dong D, Markova N, Kim PM, Ivarsson Y

Abstract
The intrinsically disordered regions of eukaryotic proteomes are enriched in short linear motifs (SLiMs), which are of crucial relevance for cellular signaling and protein regulation; many mediate interactions by providing binding sites for peptide binding domains. The vast majority of SLiMs remain to be discovered highlighting the need for experimental methods for their large-scale identification. We present a novel proteomic peptide phage display (ProP-PD) library that displays peptides representing the disordered regions of the human proteome, allowing direct large-scale interrogation of most potential binding SLiMs in the proteome. The performance of the ProP-PD library was validated through selections against SLiM-binding bait domains with distinct folds and binding preferences. The vast majority of identified binding peptides contained sequences that matched the known SLiM-binding specificities of the bait proteins. For SHANK1 PDZ, we establish a novel consensus TxF motif for its non-C-terminal ligands. The binding peptides mostly represented novel target proteins, however, several previously validated protein-protein interactions were also discovered. We determined the affinities between the VHS domain of GGA1 and three identified ligands to 40-130 μM through isothermal titration calorimetry, and confirmed interactions through co-immunoprecipitation using full-length proteins. Taken together, we outline a general pipeline for the design and construction of ProP-PD libraries and the analysis of ProP-PD derived SLiM-based protein-protein interactions. We demonstrated the methods potential to identify low affinity motif-mediated interactions for modular domains with distinct binding preferences. The approach is a highly useful complement to the current toolbox of methods for protein-protein interactions discovery. This article is protected by copyright. All rights reserved.

PMID: 28002650 [PubMed - as supplied by publisher]



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A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and N1-methyladenosine modification.

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A common class of transcripts with 5'-intron depletion, distinct early coding sequence features, and N1-methyladenosine modification.

RNA. 2016 Dec 19;:

Authors: Cenik C, Chua HN, Singh G, Akef A, Snyder MP, Palazzo AF, Moore MJ, Roth FP

Abstract
Introns are found in 5' untranslated regions (5'UTRs) for 35% of all human transcripts. These 5'UTR introns are not randomly distributed: genes that encode secreted, membrane-bound and mitochondrial proteins are less likely to have them. Curiously, transcripts lacking 5'UTR introns tend to harbor specific RNA sequence elements in their early coding regions. To model and understand the connection between coding-region sequence and 5'UTR intron status, we developed a classifier that can predict 5'UTR intron status with >80% accuracy using only sequence features in the early coding region. Thus, the classifier identifies transcripts with 5' proximal-intron-minus-like-coding regions ("5IM" transcripts). Unexpectedly, we found that the early coding sequence features defining 5IM transcripts are widespread, appearing in 21% of all human RefSeq transcripts. The 5IM class of transcripts is enriched for non-AUG start codons, more extensive secondary structure both preceding the start codon and near the 5' cap, greater dependence on eIF4E for translation, and association with ER-proximal ribosomes. 5IM transcripts are bound by the Exon Junction Complex (EJC) at non-canonical 5' proximal positions. Finally, N1-methyladenosines are specifically enriched in the early coding regions of 5IM transcripts. Taken together, our analyses point to the existence of a distinct 5IM class comprising ~20% of human transcripts. This class is defined by depletion of 5' proximal introns, presence of specific RNA sequence features associated with low translation efficiency, N1-methyladenosines in the early coding region, and enrichment for non-canonical binding by the Exon Junction Complex.

PMID: 27994090 [PubMed - as supplied by publisher]



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Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

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Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila.

Mol Syst Biol. 2016 Dec 16;12(12):893

Authors: Urbanus ML, Quaile AT, Stogios PJ, Morar M, Rao C, Di Leo R, Evdokimova E, Lam M, Oatway C, Cuff ME, Osipiuk J, Michalska K, Nocek BP, Taipale M, Savchenko A, Ensminger AW

Abstract
Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector-effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector-effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila-translocated substrates. While capturing all known examples of effector-effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct-a hallmark of an emerging class of proteins called metaeffectors, or "effectors of effectors". Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector-effector modulation, may be a common feature of many intracellular pathogens-with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.

PMID: 27986836 [PubMed - in process]



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Misregulation of an Activity-Dependent Splicing Network as a Common Mechanism Underlying Autism Spectrum Disorders.

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Misregulation of an Activity-Dependent Splicing Network as a Common Mechanism Underlying Autism Spectrum Disorders.

Mol Cell. 2016 Dec 15;64(6):1023-1034

Authors: Quesnel-Vallières M, Dargaei Z, Irimia M, Gonatopoulos-Pournatzis T, Ip JY, Wu M, Sterne-Weiler T, Nakagawa S, Woodin MA, Blencowe BJ, Cordes SP

Abstract
A key challenge in understanding and ultimately treating autism is to identify common molecular mechanisms underlying this genetically heterogeneous disorder. Transcriptomic profiling of autistic brains has revealed correlated misregulation of the neuronal splicing regulator nSR100/SRRM4 and its target microexon splicing program in more than one-third of analyzed individuals. To investigate whether nSR100 misregulation is causally linked to autism, we generated mutant mice with reduced levels of this protein and its target splicing program. Remarkably, these mice display multiple autistic-like features, including altered social behaviors, synaptic density, and signaling. Moreover, increased neuronal activity, which is often associated with autism, results in a rapid decrease in nSR100 and splicing of microexons that significantly overlap those misregulated in autistic brains. Collectively, our results provide evidence that misregulation of an nSR100-dependent splicing network controlled by changes in neuronal activity is causally linked to a substantial fraction of autism cases.

PMID: 27984743 [PubMed - in process]



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Naturally Occurring Off-Switches for CRISPR-Cas9.

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Naturally Occurring Off-Switches for CRISPR-Cas9.

Cell. 2016 Dec 15;167(7):1829-1838.e9

Authors: Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki A, Shah M, Sontheimer EJ, Maxwell KL, Davidson AR

Abstract
CRISPR-Cas9 technology would be enhanced by the ability to inhibit Cas9 function spatially, temporally, or conditionally. Previously, we discovered small proteins encoded by bacteriophages that inhibit the CRISPR-Cas systems of their host bacteria. These "anti-CRISPRs" were specific to type I CRISPR-Cas systems that do not employ the Cas9 protein. We posited that nature would also yield Cas9 inhibitors in response to the evolutionary arms race between bacteriophages and their hosts. Here, we report the discovery of three distinct families of anti-CRISPRs that specifically inhibit the CRISPR-Cas9 system of Neisseria meningitidis. We show that these proteins bind directly to N. meningitidis Cas9 (NmeCas9) and can be used as potent inhibitors of genome editing by this system in human cells. These anti-CRISPR proteins now enable "off-switches" for CRISPR-Cas9 activity and provide a genetically encodable means to inhibit CRISPR-Cas9 genome editing in eukaryotes. VIDEO ABSTRACT.

PMID: 27984730 [PubMed - in process]



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Internal Structure and Preferential Protein Binding of Colloidal Aggregates.

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Internal Structure and Preferential Protein Binding of Colloidal Aggregates.

ACS Chem Biol. 2016 Dec 16;

Authors: Duan D, Torosyan H, Elnatan D, McLaughlin CK, Logie J, Shoichet MS, Agard DA, Shoichet BK

Abstract
Colloidal aggregates of small molecules are the most common artifact in early drug discovery, sequestering and inhibiting target proteins without specificity. Understanding their structure and mechanism has been crucial to developing tools to control for, and occasionally even exploit, these particles. Unfortunately, their polydispersity and transient stability have prevented exploration of certain elementary properties, such as how they pack. Dye-stabilized colloidal aggregates exhibit enhanced homogeneity and stability when compared to conventional colloidal aggregates, enabling investigation of some of these properties. By small-angle X-ray scattering and multiangle light scattering, pair distance distribution functions suggest that the dye-stabilized colloids are filled, not hollow, spheres. Stability of the coformulated colloids enabled investigation of their preference for binding DNA, peptides, or folded proteins, and their ability to purify one from the other. The coformulated colloids showed little ability to bind DNA. Correspondingly, the colloids preferentially sequestered protein from even a 1600-fold excess of peptides that are themselves the result of a digest of the same protein. This may reflect the avidity advantage that a protein has in a surface-to-surface interaction with the colloids. For the first time, colloids could be shown to have preferences of up to 90-fold for particular proteins over others. Loaded onto the colloids, bound enzyme could be spun down, resuspended, and released back into buffer, regaining most of its activity. Implications of these observations for colloid mechanisms and utility will be considered.

PMID: 27983786 [PubMed - as supplied by publisher]



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