Covalent Protein Painting Reveals Structural Changes in the Proteome in Alzheimer Disease

Bamberger, Casimir; Pankow, Sandra; Martínez‐Bartolomé, Salvador; Ma, Michelle; Diedrich, Jolene K.; Rissman, Robert A.; Yates, John R.

doi:10.1101/2020.01.31.929117

Cited by 8 publications

(7 citation statements)

References 49 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To characterize CFTR conformations in vivo , surface exposed lysines in human bronchial epithelial cells expressing wt CFTR (HBE16o-) were covalently labeled with isotope encoded dimethyl groups directly in-dish for 10 min on ice (CPP) 7 (Fig 1A). Subsequently, cells were lysed and CFTR was co-immunoprecipitated using the CoPIT protocol 8 .…”

Section: Resultsmentioning

confidence: 99%

“…Protein structures available in PDB were visualized with JMOL (version 14.30.1) and the isosurface (isoSurface saSurface) of the epsilon amino group of lysine calculated and visualized in JMOL with a rolling probe radius of 1.2 Å. Area ratios were expressed as percentage of surface accessibility as described 7 and plotted in Prism 7 (Graphpad Inc.) Statistical analysis of area ratios per site was carried out in PCQ as described 53 . Briefly, ratio values for each lysine site were calculated with the SoPaX algorithm, which is part of ProteinClusterQuant (PCQ, https://github.com/proteomicsyates/ProteinClusterQuant), a One-way ANOVA analysis carried out and obtained results subjected to Benjamini-Hochberg correction.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Identification ofin vivoCFTR conformations during biogenesis and upon misfolding by covalent protein painting (CPP)

Pankow

Bamberger

Martínez‐Bartolomé

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

In vivo characterization of protein structures or protein structural changes after perturbation is a major challenge. Therefore, experiments to characterize protein structures are typically performed in vitro and with highly purified proteins or protein complexes. Using a novel low-resolution method named Covalent Protein Painting (CPP) that allows the characterization of protein conformations in vivo, we are the first to report how an ion channel, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), is conformationally changed during biogenesis and channel opening in the cell. Our study led to the identification of a novel opening mechanism for CFTR by revealing that the interaction of the intracellular loop 2 (ICL2) with the nucleotide binding domain 2 (NDB2) of CFTR is needed for channel gating, and this interaction occurs concomitantly with changes to the narrow part of the pore and the walker A lysine in NBD1. However, the ICL2:NBD2 interface, which forms a "ball-in-a-socket" motif, is uncoupled during biogenesis, likely to prevent inadvertent channel activation during transport. In particular, solvent accessibility of lysine 273 (K273) in ICL2 changes with the opening and closing of the channel. Mutation of K273 severely impaired CFTR biogenesis and led to accumulation of CFTR in the Golgi and TGN. CPP further revealed that, even upon treatment with current approved drugs or at permissive temperature, the uncoupled state of ICL2 is a prominent feature of the misfolded CFTR mutants ΔF508 and N1303K that cause Cystic Fibrosis (CF), which suggests that stabilization of this interface could produce a more efficient CF drug. CPP is able to characterize a protein in its native environment and measure the effect of complex PTMs and protein interactions on protein structure, making it broadly applicable and invaluable for the development of new therapies.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Identification ofin vivoCFTR conformations during biogenesis and upon misfolding by covalent protein painting (CPP)

Pankow

Bamberger

Martínez‐Bartolomé

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…3b-c). Besides the relatively wide coverage 24 , distribution plot of the labeled lysine residues further shows the labeling reaction exploited by TRAP is not preferential towards any secondary structure elements (Fig. 2d, Extended Data Fig.…”

Section: A Landscape Of Glycolytic Targetome In Human Cancer Cellsmentioning

confidence: 93%

Chemoproteomics Maps Glycolytic Targetome in Cancer Cells

Hao

Tian

Wan

et al. 2021

Preprint

View full text Add to dashboard Cite

Hyperactivated glycolysis, favoring uncontrolled growth and metastasis by producing essential metabolic intermediates engaging bioenergetics and biosynthesis, is a metabolic hallmark of most cancer cells. Although sporadic information has revealed glycolytic metabolites also possess non-metabolic function as signaling molecules, it remains largely elusive how these metabolites interact with and functionally regulate their binding targets. Here we introduce a Target Responsive Accessibility Profiling (TRAP) approach that measures ligand binding-induced steric hindrance in protein targets via global profiling accessibility changes in reactive lysines, and mapped 913 target candidates and 2,487 interactions for 10 major glycolytic metabolites in cancer cells via TRAP. The elucidated targetome uncovers diverse regulatory modalities of glycolytic metabolites involving the direct perturbation of carbohydrate metabolism enzymes, intervention of transcriptional control, modulation of proteome-level acetylation and protein complex assemblies. The advantages gained from glycolysis by cancer cells are expanded by discovering lactate as a ligand for an orphan transcriptional regulator TRIM 28 that promotes p53 degradation, and by identifying pyruvate acting against a cell apoptosis inducer trichostatin A via attenuating protein acetylation. Lastly, the inhibition of glycolytic key enzymes led to identify an intrinsically active glycolytic intermediate glyceraldehyde 3-phosphate that elicits its cytotoxicity by engaging with ENO1 and MTHFD1. Collectively, the glycolytic targetome depicted by TRAP constitutes a fertile resource for understanding how glycolysis finely tunes metabolism and signaling in support of cancer cells, and fostering the exploitation of glycolytic targetome as promising nodes for anti-cancer therapeutics development.

show abstract

“…3b-c ). Besides the relatively wide coverage 21 , distribution plot of the labeled lysine residues further shows the labeling reaction exploited by TRAP is not preferential towards any secondary structure elements ( Fig. 2d, Extended Data Fig.…”

Section: A Landscape Of Glycolytic Targetome In Human Cancer Cellsmentioning

confidence: 93%

Chemoproteomics Maps Glycolytic Targetome in Cancer Cells

Tian¹,

Wan²,

Ding

et al. 2020

Preprint

View full text Add to dashboard Cite

Hyperactivated glycolysis, favoring uncontrolled growth and metastasis by producing essential metabolic intermediates engaging bioenergetics and biosynthesis, is a metabolic hallmark of most cancers. Although sporadic information has revealed glycolytic metabolites also possess non-metabolic function as signaling molecules, it remains largely elusive how these metabolites interact and functionally regulate their binding targets. Here, we developed a Target Responsive Accessibility Profiling (TRAP) approach for mapping the glycolytic targetome in cancer cells. We identified 913 proteins and 2,487 interactions as target candidates for 10 metabolites involved in glycolysis. The elucidated targetome uncovers diverse regulatory modalities of glycolytic metabolites involving the direct perturbation of carbohydrate metabolism enzymes, intervention of transcriptional control, modulation of proteome-level acetylation and disruption of protein complex assemblies. The advantages gained by enhanced glycolysis in cancer cells through these distinct mechanisms are innovated by discovering lactate as a ligand for an orphan transcriptional regulator TRIM 28 that promotes p53 degradation, and by identifying pyruvate acting against a cell apoptosis inducer trichostatin A via attenuating protein acetylation. Lastly, the inhibition of glycolytic key enzymes led to identify an intrinsically active glycolytic intermediate, glyceraldehyde 3-phosphate, that elicits its cytotoxic effects by engaging with ENO1 and MTHFD1. Collectively, the mapped targetome constitutes a fertile source for understanding how glycolysis finely tunes cancer metabolism and signaling in favor of tumor growth, and fostering the exploitation of glycolytic targetome as promising nodes for cancer therapeutics development.

show abstract

Covalent Protein Painting Reveals Structural Changes in the Proteome in Alzheimer Disease

Cited by 8 publications

References 49 publications

Identification ofin vivoCFTR conformations during biogenesis and upon misfolding by covalent protein painting (CPP)

Identification ofin vivoCFTR conformations during biogenesis and upon misfolding by covalent protein painting (CPP)

Chemoproteomics Maps Glycolytic Targetome in Cancer Cells

Chemoproteomics Maps Glycolytic Targetome in Cancer Cells

Contact Info

Product

Resources

About