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Mapping Myc Interactome by Micro-Affinity Purification Coupling Mass Spectrometry

ABSTRACT: Affinity purification-mass spectrometry (AP-MS) is a powerful approach for protein-protein interaction (PPI) analysis. Here, we report the development of a micro-scale immobilized metal affinity chromatography (m-IMAC) platform for rapid and reproducible purification of protein complexes prior to mass spectrometric analysis. The method was applied for mapping PPIs of Myc protein which regulates the expression of thousands of genes and plays a central role in tumorigenesis. Systematic characterization of Myc interactome is essential for understanding how a single transcription factor orchestrates such diverse biological processes. Affinity purification was performed using m-IMAC columns fabricated by photoinitiated polymerization within pipette tips (spin-tip columns), enabling efficient enrichment of Myc interacting proteins. Analysis with Label-Free Quantification (LFQ) with Data-Independent Acquisition (DIA) proteomics identified 574 Myc interacting proteins, the majority of which have not been previously reported as the Myc interactors. Two newly identified Myc associated factors were validated by biochemical assays, and the overall dataset showed good concordance with the reported Myc interactors. Gene Ontology (GO) enrichment analysis of the Myc associated proteome revealed significant enrichment in macromolecular metabolism (particularly RNA processing), regulation of gene expression, chromatin organization and remodeling. This work provides new insights into Myc mediated transcriptional regulation in proteomic level. Importantly, owing to its straightforward, rapid operation, the m-IMAC platform provides a general and efficient analytical strategy for high-throughput interactome analysis.

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ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: ying zhu

LAB HEAD: Jingwu Kang

PROVIDER: PXD073401 | Pride | 2026-06-09

REPOSITORIES: Pride

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Project description:Liposomal amphotericin B is an important frontline drug for the treatment of visceral leishmaniasis, a neglected disease of poverty. The mechanism of action of amphotericin B (AmB) is thought to involve interaction with ergosterol and other ergostane sterols, resulting in disruption of the integrity and key functions of the plasma membrane. Emergence of clinically refractory isolates of L. donovani and L. infantum is an ongoing issue and knowledge of potential resistance mechanisms can help to alleviate this problem. Here we report the characterisation of four independently selected L. donovani clones that are resistant to AmB. Whole genome sequencing revealed that in three of the moderately resistant clones, resistance was due solely to the deletion of a gene encoding C24-sterol methyltransferase (SMT1). The fourth, hyper-resistant resistant clone (>60-fold) was found to have a 24 bp deletion in both alleles of a gene encoding a putative cytochrome P450 reductase (P450R1). Metabolic profiling indicated these parasites were virtually devoid of ergosterol (0.2% versus 18% of total sterols in wild-type) and had a marked accumulation of 14-methylfecosterol (75% versus 0.1% of total sterols in wild-type) and other 14-alpha methylcholestanes. These are substrates for sterol 14-alpha demethylase (CYP51) suggesting that this enzyme is a bona fide P450R specifically involved in electron transfer from NADPH to CYP51 during catalysis. Deletion of P450R1 in wild-type cells phenocopied the metabolic changes observed in our AmB hyper-resistant clone as well as in CYP51 nulls. Likewise, addition of a wild type P450R1 gene restored sterol profiles to wild type. Our studies indicate that P450R1 is essential for L. donovani amastigote viability, thus loss of this gene is unlikely to be a driver of clinical resistance. Nevertheless, investigating the mechanisms underpinning AmB resistance in these cells provided insights that refine our understanding of the L. donovani sterol biosynthetic pathway.

			Action	DRS
	Astral_14min_A1.raw	Raw
	Astral_14min_A2.raw	Raw
	Astral_14min_A3.raw	Raw
	Astral_14min_G1.raw	Raw
	Astral_14min_G2.raw	Raw

Dataset Information

Mapping Myc Interactome by Micro-Affinity Purification Coupling Mass Spectrometry

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