Dataset Information

Functional and structural characterization of zebrafish ASC.

ABSTRACT: The zebrafish genome encodes homologs for most of the proteins involved in inflammatory pathways; however, the molecular components and activation mechanisms of fish inflammasomes are largely unknown. ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)] is the only adaptor involved in the formation of multiple types of inflammasomes. Here, we demonstrate that zASC is also involved in inflammasome activation in zebrafish. When overexpressed in vitro and in vivo in zebrafish, both the zASC and zASC pyrin domain (PYD) proteins form speck and filament structures. Importantly, the crystal structures of the N-terminal PYD and C-terminal CARD of zebrafish ASC were determined independently as two separate entities fused to maltose-binding protein. Structure-guided mutagenesis revealed the functional relevance of the PYD hydrophilic surface found in the crystal lattice. Finally, the fish caspase-1 homolog Caspy, but not the caspase-4/11 homolog Caspy2, interacts with zASC through homotypic PYD-PYD interactions, which differ from those in mammals. These observations establish the conserved and unique structural/functional features of the zASC-dependent inflammasome pathway.

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Structural data are available in the PDB under accession numbers 5GPP and 5GPQ.

SUBMITTER: Li Y

PROVIDER: S-EPMC6105367 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Functional and structural characterization of zebrafish ASC.

Li Yajuan Y Huang Yi Y Cao Xiaocong X Yin Xueying X Jin Xiangyu X Liu Sheng S Jiang Jiansheng J Jiang Wei W Xiao Tsan Sam TS Zhou Rongbin R Cai Gang G Hu Bing B Jin Tengchuan T

The FEBS journal 20180614 14

The zebrafish genome encodes homologs for most of the proteins involved in inflammatory pathways; however, the molecular components and activation mechanisms of fish inflammasomes are largely unknown. ASC [apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD)] is the only adaptor involved in the formation of multiple types of inflammasomes. Here, we demonstrate that zASC is also involved in inflammasome activation in zebrafish. When overexpressed in vitro and in ...[more]

PMID: 29791979

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Project description:IntroductionZebrafish regenerate their retinas following damage, resulting in restoration of visual function. Here we evaluate recovery of retinal function through qualitative and quantitative analysis of the electroretinogram (ERG) over time following retinal damage, in correlation to histological features of regenerated retinal tissue.MethodsRetinas of adult zebrafish were lesioned by intravitreal injection of 10 μM (extensive lesion; destroys all neurons) or 2 μM (selective lesion; spares photoreceptors) ouabain. Unlesioned contralateral retinas served as controls. Function of retinal circuitry was analyzed at selected timepoints using ERG recordings from live zebrafish, and whole eyes were processed for histological analyses immediately thereafter.ResultsQualitative and quantitative assessment of waveforms during retinal regeneration revealed dynamic changes that were heterogeneous on an individual level within each sampling time, but still followed common waveform recovery patterns on a per-fish and population-level basis. Early in the regeneration period (13-30 days post injury; DPI), for both lesion types, b-waves were essentially not detected, and unmasked increased apparent amplitudes, implicit times, and half-widths of a-waves (vs. controls). In control recordings, d-waves were not obviously detected, but apparent d-waves (OFF-bipolar responses) from regenerating retinas of several fish became prominent by 30DPI and dominated the post-photoreceptor response (PPR). Beyond 45DPI, b-waves became detectable, and the ratio of apparent d- to b-wave contributions progressively shifted with most, but not all, fish displaying a b-wave dominated PPR. At the latest timepoints (extensive, 90DPI; selective, 80DPI), recordings with measurable b-waves approached a normal waveform (implicit times and half-widths), but amplitudes were not restored to control levels. Histological analyses of the retinas from which ERGs were recorded showed that as regeneration progressed, PKCa + ON-bipolar terminals and parvalbumin + amacrine cell processes became more stereotypically positioned within the deep sublaminae of the INL over recovery time after each lesion type, consistent with the shift in PPR seen in the ERG recordings.DiscussionTaken together, these data suggest that photoreceptor-OFF-bipolar component/connectivity may functionally recover and mature earlier during regeneration compared to the photoreceptor-ON-bipolar component, though the timeframe in which such recovery happens is heterogeneous on a per-fish basis. Collectively our studies suggest gradual restoration of ON-bipolar functional circuitry during retinal regeneration.

Project description:The spliceosome is a dynamic macromolecular machine that catalyzes the removal of introns from pre-mRNA to make mature message. Schizosaccharomyces pombe Cwf10 (homolog Saccharomyces cerevisiae Snu114 and of Human U5-116K), an integral member of the U5 snRNP, is a GTPase that shares sequence homology with the eukaryotic translation elongation factor EF2. Cwf10 is required for pre-mRNA splicing; however, its mechanism(s) of action is still not understood. Cwf10/Snu114 family members contain a conserved N-terminal extension (NTE) that lacks homology with EF2 and has been predicted to be an intrinsically unfolded domain. Using S. pombe as a model system, we show that the NTE is not essential, but cells lacking this domain are defective in pre-mRNA splicing at all temperatures. Genetic interactions between cwf10-M-NM-^TNTE and other pre-mRNA splicing mutants are consistent with a role for the NTE in spliceosome activation. Characterization of Cwf10-NTE by various biophysical techniques shows the NTE contains both regions of structure and disorder in solution. The first twenty-three highly-conserved amino acids of the NTE are essential for its role in splicing, but are not sufficient to restore pre-mRNA splicing to wild-type levels in cwf10-M-bM-^HM-^FNTE cells. When the NTE is overexpressed in the cwf10-M-NM-^TNTE background, it can complement the truncated Cwf10 protein in trans, and it also immunoprecipitates a complex similar in composition to the late-stage U5.U2/U6 spliceosome. These data show that the structurally flexible NTE is capable of making specific contacts within the spliceosome that may facilitate Cwf10M-bM-^@M-^Ys overall role facilitating spliceosome rearrangements. Interrogation of the S. pombe transcriptome using poly-A enriched RNA sequencing (Illumina HiSeq 2500) in wild type and cwf10-M-NM-^TNTE cultures. A total of 4 samples were analysed: two biological repeats of wild-type strain and two biological repeats of cwf10-M-NM-^TNTE

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Functional and structural characterization of zebrafish ASC.

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Functional and structural characterization of zebrafish ASC.

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