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Mapping the Binding Interactions Between human Gasdermin D and human Caspase-1 Using Carbene Footprinting

ABSTRACT: Carbene footprinting is a recently developed mass spectrometry-based chemical labeling technique that probes protein interactions and conformation. Here, we use the methodology to investigate binding interactions between the protease human Caspase-1 (C285A) and full-length human Gasdermin D (hGSDMD), which are important in inflammatory cell death. GSDMD is cleaved by Caspase-1, releasing its N-terminal domain which oligomerizes in the membrane to form large pores, resulting in lytic cell death. Regions of reduced carbene labeling (masking), caused by protein binding, were observed for each partner in the presence of the other and were consistent with hCaspase-1 exosite and active-site interactions. Most notably, the results showed direct occupancy of hCaspase-1 active site by hGSDMD for the first time. Differential carbene labeling of full-length hGSDMD and the pore-forming N-terminal domain assembled in liposomes showed masking of the latter consistent with oligomeric assembly and insertion into the lipid bilayer. Interactions between Caspase-1 and the specific inhibitor VRT-043198 were also studied by this approach. In wild-type Caspase-1, VRT-043198 alkylates the active-site C285. Here we showed by carbene labeling that this inhibitor can occupy the active-site of a C285A mutant reversibly. These findings add considerably to our knowledge of the hCaspase-1-hGSDMD system.

INSTRUMENT(S): LTQ FT

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: James Lloyd

LAB HEAD: Neil Oldham

PROVIDER: PXD040212 | Pride | 2023-08-14

REPOSITORIES: Pride

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Project description:Background: With lower manufacturing cost, high spot density, and flexible probe design, genomic tiling microarrays are ideal for comprehensive transcriptome studies. Typically, transcriptome profiling using microarrays involves reverse transcription, which converts RNA to cDNA. The cDNA is then labeled and hybridized to the probes on the arrays, thus the RNA signals are detected indirectly. Reverse transcription is known to generate artifactual cDNA, in particular the synthesis of second-strand cDNA, leading to false discovery of antisense RNA. To address this issue, we have developed an effective method using RNA that is directly labeled, thus by-passing the cDNA generation. This paper describes the development of this method and its application to mapping transcriptome profiles. Results: RNA extracted from laboratory cultures of Porphyromonas gingivalis was fluorescently labeled with an alkylation reagent and hybridized directly to probes on genomic tiling microarrays specifically designed for this periodontal pathogen. The generated transcriptome profile was strand-specific and produced signals close to background level in most antisense regions of the genome. In contrast, high levels of signal were detected in the antisense regions when the hybridization was done with cDNA. In addition, five antisense areas were tested with independent strand-specific RT-PCR and none to negligible amplification was detected, indicating that the strong antisense cDNA signals were artifacts. Conclusions: An efficient method was developed for mapping transcriptome profiles specific to both coding strands of a bacterial genome. This method chemically labels and uses extracted RNA directly in microarray hybridization. The generated transcriptome profile was free of cDNA artifactual signals. In addition, this method requires fewer processing steps and is more sensitive in detecting small amount of RNA compared to end-labeling methods due to the incorporation of more fluorescent molecules per RNA fragment. This serial of experiments were performed to compare the transcriptome profiles revealed between the use of cDNA (converted from RNA) and RNA. Three samples of cDNA-based method were provided and two samples of RNA-based method were included. The signal intensities of these arrays were normalized based on both the in-between array method and the genomic DNA reference arrays (included) using the R 'tilingarray' package and these experiments were referred in the paper.

Dataset Information

Mapping the Binding Interactions Between human Gasdermin D and human Caspase-1 Using Carbene Footprinting

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