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Confirmation of the phosphorylation sites of human PhK

ABSTRACT: The activity of PhK is regulated by phosphorylation, and we utilized single-particle cryo-electron microscopy to solve the high-resolution structures of human PhK under phosphorylation and calcium ion activation. To gain a deeper insight into the phosphorylation sites following PKA treatment, we employed tandem mass spectrometry for precise localization of the phosphorylation sites within PhK.

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Ruifang Ma

LAB HEAD: Kaige Yan

PROVIDER: PXD059345 | Pride | 2025-05-07

REPOSITORIES: Pride

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			Action	DRS
	2E.raw	Raw
	2E_modificaition_site.xlsx	Xlsx
	2E_peptide_view.xlsx	Xlsx
	2E_protein_view.xlsx	Xlsx
	M_1.raw	Raw

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Publications

Molecular basis for the regulation of human phosphorylase kinase by phosphorylation and Ca<sup>2</sup>.

Ma Ruifang R Du Bowen B Shi Chen C Wang Lei L Zeng Fuxing F Han Jie J Guan Huiyi H Wang Yong Y Yan Kaige K

Nature communications 20250328 1

Phosphorylase kinase (PhK) regulates the degradation of glycogen by integrating diverse signals, providing energy to the organism. Dysfunctional mutations may directly lead to Glycogen Storage Disease type IX (GSD IX), whereas the abnormal expression of PhK is also associated with tumors. Here, we use cryo-electron microscopy (cryo-EM) to resolve its near-atomic structures in the inactive and active states. These structures reveal the interactions and relative locations of the four subunits (αβγ ...[more]

PMID: 40148320

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Confirmation of the binding site of spermine to ATP13A2 protein

Project description:To better understand human ATP13A2-mediated polyamine transport, we used single-particle cryo-electron microscopy to solve high-resolution structures of human ATP13A2. Interestingly, we found polyamine binding at multiple sites distributed along the transmembrane regions in ATP13A2 protein, which has not been reported in previous studies. Therefore, we used cross-linking mass spectrometry (CX-MS) to confirm potential binding sites.

2023-05-10 | PXD037493 | Pride

Proteome and phosphoproteome in sh-HNRNPA2B1-8226 and sh-NC-8226

Project description:Utilizing 4D label-free LC/MS, we conducted a comprehensive analysis of the quantitative proteome and phosphoproteome in MM cells, comparing those transfected with sh-HNRNPA2B1-8226 against control sh-NC-8226.

2025-11-24 | PXD057824 | Pride

Global ovary gene interaction network structure informs biological effects of Chemical stressors

Project description:Background: Complex biological networks control fundamental processes such as reproduction. The relationship of network structure to biological function was investigated in a global gene interaction network developed from ovary tissues of the model fish, fathead minnow (Pimephales promelas). Results: A global ovary gene interaction network was inferred from gene expression in ovaries of fathead minnow representing 288 different exposure conditions and 1,472 microarrays. More than 74 percent of the interactions in two subnetworks and 37% of the connections in a third subnetwork were also present as known connections found in curated databases and the literature. Subnetworks within the network were enriched in specific pathways and key ovarian functions. The network location of differentially expressed genes from different ovary stages was consistent with known functional changes at those stages. The global network provide additional insight into gene function when gene directly linked to differentially expressed genes are considered in enrichment analysis. Analysis of the impact of bisphenol A on ovarian gene expression demonstrated that the network provides an informative frame work in which to investigate mechanisms by which chemical effects occur. Conclusions: Our results suggest that the ovary transcriptional network is composed of several connected subnetworks where each is enriched in specific functions. Construction of a global gene regulatory network from multiple experiments provides valuable insight into the function of genes and the impact of chemicals on biological systems. total RNA from the ovary tissue of treated or control fish labeled in single color was hybridized to Agilent fathead minnow microarray (design 019597)

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A Network G Protein Gai Signaling Partners is Revealed by Proximity Labeling Proteomics and Includes PDZ-RhoGEF

Project description:G protein-coupled receptors (GPCRs) that couple to the Gi family of G proteins are key regulators of cell and tissue physiology. Our previous work has revealed new roles for Gαi in regulating the migration of neutrophils and fibrosarcoma cells downstream of activated chemoattractant receptors. We used an intact cell proximity-based labeling approach using BioID2 coupled to tandem mass tag (TMT)-based quantitative proteomics to identify proteins that selectively interacted with the GTP-bound form of Gαi1. Multiple targets were identified and validated for selective biotinylation by a constitutively active BioID2-tagged Gai1 mutant, suggesting a network of interactions for activated Gαi proteins in intact cells. We showed that active Gαi1 stimulated one candidate protein, PDZ‐RhoGEF (PRG) and that despite over 85% sequence identity, the ability of Gαi1, Gαi2, and Gαi3 isoforms to activate PRG greatly differed. We also demonstrated that active Gαi likely regulates polarized myosin light chain kinase phosphorylation through activation of PRG in primary human neutrophils, suggesting functional relevance of this interaction. Identification and characterization of new targets regulated by Gαi both individually and in networks provide insights that will aid in the investigation of the functional roles of Gi-coupled GPCRs in multiple biological processes.

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OTC variant confirmation

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Transcriptome-proteome profiling in Burkholderia thailandensis during the transition from exponential to stationary phase

Project description:Burkholderia thailandensis is closely related to highly pathogenic Burkholderia species, and it is an excellent surrogate as it rarely causes disease in humans. Understanding the complex molecular mechanisms that characterize the transition of B. thailandensis from exponential to stationary phases is critical to understanding bacterial responses to stress or nutrient limitation. We present here an integrated transcriptomic and proteomic analysis to profile gene and protein expression changes during entry into stationary phase. We identified 928 differentially expressed genes (DEGs) and 832 differentially expressed proteins (DEPs), highlighting significant transcriptional and translational reprogramming. Genes encoding proteins involved in benzoate degradation and O-antigen nucleotide sugar biosynthesis were among the most highly upregulated in stationary phase, whereas processes such as nitrogen metabolism, translation, and flagellar biosynthesis were downregulated. At the proteome level, proteins related to fatty acid degradation and butanoate metabolism accumulated along with proteins involved in synthesis of secondary metabolites. Markedly downregulated proteins included ribosomal proteins and translation factors as well as the house-keeping iron-sulfur biogenesis proteins. A protein-protein interaction (PPI) network analysis identified clusters involved in processes such as fatty acid metabolism and amino acid degradation. Surprisingly, the RpoS sigma factor, which has been shown to accumulate in stationary phase in several bacterial species, was not significantly increased in B. thailandensis during stationary phase. These findings offer comprehensive insights into B. thailandensis adaptive strategies and provide a foundation for developing interventions against pathogenic Burkholderia species.

2025-07-16 | PXD056625 | Pride

Cryo-EM structure of the human COP1-DET1 ubiquitin ligase complex

Project description:Ubiquitin modification regulates fundamental cellular processes by controlling protein stability and function. The ubiquitin ligase COP1, conserved from plants to humans, targets developmental transcription factors. COP1 can act on its own or be incorporated into CULLIN4–RING ligase (CRL4) complexes via the adaptor DET1. Despite its importance, the architecture and mechanisms of COP1- and DET1-containing assemblies remain incompletely defined. Here, we present cryo-EM structures of human COP1 bound to DDB1–DDA1–DET1 (DDD) and Ube2e2, capturing an inactive, stacked assembly. Co-expression with COP1 substrates such as c-Jun or ETS2 disrupts this arrangement and drives a conformational transition into a distinct dimeric state that permits substrate access. Structural modeling delineates the spatial organization of the COP1 WD40 domains responsible for substrate recruitment. DET1 functions as a scaffold, linking COP1 and Ube2e2 to initiate ubiquitin transfer to substrates, while DDB1 recruits the CULLIN4–RBX1 complex to enable Ube2d3-mediated ubiquitin chain elongation. Together, these findings reveal dynamic switching between structural states of the CRL4DET1–COP1 E3 ligase and a substrate-triggered activation mechanism, providing a framework for understanding substrate-specific regulation and E3 ligase assembly dynamics.

2026-01-23 | PXD071633 | Pride

Human immunoglobulin heavy gamma chain polymorphisms: molecular confirmation of proteomic assessment

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Confirmation of SOMAmer® Specificity in Serum using Mass Spectrometry

Project description:Experiments were conducted to confirm SOMAmer® reagent specificity by enrichment in serum from elderly subjects (>65 years, male n=1 and female n=1, purchased from commercial vendor) followed by measurement using data dependent analysis (DDA). Non-specific binding was assessed in the biological matrix by comparing target protein spectral counts in the respective enrichment to pull down using a reagent generated against the bacterial protein CysH. A positive hit was defined as target protein detection with a minimum of 2 spectral counts and signal over CysH background greater than 10X (if applicable).

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Kinase-catalyzed Biotinylation to Map Cell Signaling Pathways (K-BMAPS): Application to Epidermal Growth Factor Signaling

Project description:Cell signaling involves a network of protein-protein interactions and post-translational modifications that govern cellular responses to environmental cues. To understand and ultimately modulate these signaling pathways to confront disease, the complex web of proteins that becomes phosphorylated after extracellular stimulation has been studied using mass spectrometry-based proteomics methods. To complement prior work and fully characterize all phosphorylated proteins after stimulation of cell signaling, we developed K-BMAPS (Kinase-catalyzed Biotinylation to MAP Signaling), which utilizes ATP-biotin as a kinase cosubstrate to biotin label substrates. As a first application of K-BMAPS, the well-characterized epidermal growth factor receptor (EGFR) kinase signaling pathway was monitored by treating EGF-stimulated HeLa lysates with ATP-biotin, following by streptavidin enrichment and quantitative mass spectrometry analysis. Based on the dynamic phosphoproteins identified, a pathway map was developed considering functional categories and known interactors of EGFR. Remarkably, 94% of K-BMAPS hit proteins were included in the EGFR pathway map. With many proteins involved in transcription, translation, cell adhesion, and GTPase signaling, K-BMAPS identified phosphoproteins associated with late and continuous signaling events. In summary, K-BMAPS is a powerful tool to map the dynamic phosphorylation governing cell signaling pathways.

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