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Potential binding partners of profilin protein from Leishmania donovani

ABSTRACT: For identifying potential ligands of Leishmania profilin (LdPfn) protein, full length profilin was cloned and expressed with GST tag. Only GST tag protein was used as a negative control. Employing pull down assay and with the help of LC-MS, the potential binding partners of profilin protein in Leishmania promastigotes were detected.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Leishmania Donovani Bpk282a1

TISSUE(S): Promastigote

DISEASE(S): Visceral Leishmaniasis

SUBMITTER: Purna Sudha Bindu Ambaru

LAB HEAD: Chhitar. M. Gupta

PROVIDER: PXD026036 | Pride | 2022-03-10

REPOSITORIES: Pride

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			Action	DRS
	GST_profilin_replicate_1.raw	Raw
	GST_profilin_replicate_1.xlsx	Xlsx
	GST_profilin_replicate_2.raw	Raw
	GST_profilin_replicate_2.xlsx	Xlsx
	GST_profilin_replicate_3.raw	Raw

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Project description:Leishmania donovani, an intracellular protozoan parasite, is the causative agent of visceral leishmaniasis or kala-azar, the most severe form of leishmaniasis in humans. To date, our understanding of the molecular mechanisms associated with the pathogenicity of Leishmania infection is still limited. RNA interference—collectively RNA silencing pathways—participates in the regulation of various biological processes in most eukaryotic cells. Complexes of Argonaute proteins with small RNAs are core components of the RNA interference system and play a key role in silencing gene expression. It is becoming increasingly clear that several intracellular pathogens target host cell RNA interference pathways to promote their survival. In this study, we investigated the potential role of host macrophage Argonautes in Leishmania pathogenesis. Western blot analysis showed that protein abundance of infected macrophage Argonaute 1 (Ago1) was selectively and significantly higher than that of non-infected control at 24 h post-infection, suggesting that Ago1 plays a role in pathogenicity. In fact, siRNA-mediated downregulation of Ago1 enhanced Leishmania clearance from infected host cells, linking macrophage Ago1 to Leishmania virulence. To investigate the mechanisms of host Ago1 in Leishmania pathogenesis, a stable isotope labeling by amino acids in cell culture (SILAC)-based whole proteome approach was employed, which showed that expression of several previously reported Leishmania pathogenesis-related proteins were dependent on the level of macrophage Ago1. Moreover, the proteomic-based detailed biochemical analysis showed that Leishmania modulated host RNA-induced silencing complex (RISC) composition during infection, strongly suggesting macrophage RISC targeting. Strikingly, Leishmania proteins were detected as part of host RISC in infected cells. Together, our results demonstrate that Leishmania targets host RNA interference machinery to promote its survival inside the host macrophage.

Project description:Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway. For microarray screening, Invitrogen Protoarray v4.0 protein microarrays were used. Human NEMO expressed as a C-terminal GST fusion was purified from E. coli lysates and labelled with biotin. NEMO or biotinylated GST were applied to the microarrays and binding partners detected using streptavidin-Alexa Fluor 647. Significant interactors on both arrays were detected using Invitrogen Protoarray Prospector software and a Z-score cutoff of 3.0. Following subtraction of interactors present on the GST control array, a final set of significant NEMO interactors was derived. Full experimental details are supplied in Fenner, B. J., Scannell, M. & Prehn, J. H. M. (2010). Expanding the substantial interactome of NEMO using protein microarrays. PLoS ONE (in press).

Project description:Transcriptome analysis via high-throughput sequencing (RNA-Seq) captures significant changes in gene expression. The purpose of this study is to elucidate the transcriptomic changes in profilin deficient Leishmania promastigotes (LdPfn+/-) as compared to wild type control (LdPfn+/+). This research will enable us to gain insight into the role of profilin in Leishmania cells. The data has been validated by qRT-PCR. Data analysis results are further supported by flow cytometry, immunofluorescence experiments and by western blot demonstrating a depletion in the protein expression of a eukaryotic translation initiation factor in the cells with depleted levels of profilin protein. Methods: Total RNA from three independent biological replicates from each of wild-type (LdPfn+/+) and profilin knockout (LdPfn+/-) Leishmania promastigotes was extracted and after library preparation, RNA sequencing was carried out on Illumina HiSeq 2500. The sequence reads that passed quality filters were aligned to the Leishmania donovani (BPK282A1) genomic data obtained from TriTrypDB version 51 using Bowtie2 (-x option). All analyses were carried out using the Tophat pipeline with the following versions: Tophat v2.1.1, Bowtie2 v2.3.5.1. The HTSeq version 0.12.4 (htseq-count -f option) was used to count the number of reads aligned to protein-coding genes. DeSeq tool was used for differential gene expression analysis between samples in protein-coding genes. qRT-PCR validation was performed using SYBR Green assay. Results: RNA-Seq datasets generated in this study with each sample control (LdPfn+/+) and profilin knockout (LdPfn+/-) have at least 30 million read pairs. RNA-seq data were aligned to the L.donovani genome (Leishmania donovani BPK282A1, NCBI taxon ID: 981087) and 8135 transcripts were identified in our data set. Data analysis revealed that a total of 254 genes were differentially expressed (DE) having a p-value <0.05. The data obtained from RNA-Seq have been validated by real-time PCR of 18 genes, 8 UP regulated and 10 down-regulated. Results of qRT-PCR analysis showed a strong correlation with the RNA-seq data. Inferences drawn from transcriptomic analysis were further validated by flow cytometry, immunofluorescence experiments and western blotting, revealing a critical role during the early phases of Leishmania cell division. Conclusions: Present study represents a comprehensive report elucidating the transcriptomic changes in profilin deficient Leishmania promastigotes (LdPfn+/-) as compared to wild type (LdPfn+/+). This RNA-Seq data along with validation by qPCR, flow cytometry experiments, and immunofluorescent microscopy experiments indicate that profilin might play a critical role during the early phases of Leishmania cell division.

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Potential binding partners of profilin protein from Leishmania donovani

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