<scp>PyXlinkViewer</scp>: A flexible tool for visualization of protein chemical crosslinking data within the <scp>PyMOL</scp> molecular graphics system

Pannexin 1 (PANX1) is a glycoprotein that forms large pore channels capable of passing ions and metabolites such as ATP for cellular communication. PANX1 has been implicated in many diseases including breast cancer and melanoma, where inhibition or deletion of PANX1 reduced the tumorigenic and metastatic properties of the cancer cells. We interrogated the effect of single amino acid changes in various PANX1 domains, using naturally occurring variants reported in cancer patient tumors. We found that a previously reported variant (Q5H), is present in cancer cells, but was not different from the wildtype (Q5) in glycosylation, trafficking, or channel function, and did not affect cellular properties. We discovered that the Q5H variant is in fact the highly conserved ancestral allele of PANX1, with 89% of humans carrying at least one Q5H allele. Another mutated form Y150F, found in a melanoma patient tumour, prevented phosphorylation at Y150 as well as complex N-glycosylation, while increasing intracellular localization. SRC is the predicted kinase to phosphorylate the Y150 residue, and its phosphorylation is not likely to be constitutive, but rather dynamically regulated. The Y150 phosphorylation site is the first one reported to play a role in regulating post-translational modifications and trafficking of PANX1, with potential consequences on its large-pore channel structure and function in melanoma cells.

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“…Sequence alignment and superposition were performed using Pymol ( Alexander et al. , 2011 ; Schiffrin et al. , 2020 ) .…”

Section: Methodsmentioning

confidence: 99%

Pannexin 1 mutation found in melanoma tumor reduces phosphorylation, glycosylation, and trafficking of the channel-forming protein

Nouri‐Nejad

O'Donnell

Patil

et al. 2021

MBoC

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“…Across different species and proteins, the SBC motif is highly conserved in sequence and in structure, with the nonpolar residue (Φ in the Φ-π-S-S/T-S/T SBC motif) surrounded by aromatic residues. All molecular representations were generated using PyMOL 65 .…”

Section: Methodsmentioning

confidence: 99%

SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition

Shi

Cui

et al. 2021

Nat Commun

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Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.

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“…Residues with a cut-off score above 120 were mapped on to a sequence alignment (by Clustal Omega) with EphA2 to identify the equivalent residues. The visualisation was achieved using the PyXlinkViewer [ 35 ] Pymol plug-in, where the EphA2 crystal structure (PDB: 7KJB) was used as a model to represent the EphB6 cross-linking events.…”

Section: Methodsmentioning

confidence: 99%

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs

Liang

Roy

Horne

et al. 2021

Biochemical Journal

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EphB6 and EphA10 are two poorly characterised pseudokinase members of the Eph receptor family, which collectively serves as mediators of contact-dependent cell-cell communication to transmit extracellular cues into intracellular signals. As per their active counterparts, EphB6 and EphA10 deregulation is strongly linked to proliferative diseases. However, unlike active Eph receptors, whose catalytic activities are thought to initiate an intracellular signalling cascade, EphB6 and EphA10 are classified as catalytically-dead, raising the question of how non-catalytic functions contribute to Eph receptor signalling homeostasis. In this study, we have characterised the biochemical properties and topology of the EphB6 and EphA10 intracellular regions comprising the juxtamembrane region, pseudokinase and SAM domains. Using small-angle X-ray scattering and crosslinking-mass spectrometry, we observed high flexibility within their intracellular regions in solution and a propensity for interaction between the component domains. We identified tyrosines in the juxtamembrane region of EphB6 as EphB4 substrates, which can bind the SH2 domains of signalling effectors, including Abl, Src and Vav3, consistent with cellular roles in recruiting these proteins for downstream signaling. Furthermore, our finding that EphB6 and EphA10 can bind ATP and ATP-competitive small molecules raises the prospect that these pseudokinase domains could be pharmacologically-targeted to counter oncogenic signalling.

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PyXlinkViewer: A flexible tool for visualization of protein chemical crosslinking data within the PyMOL molecular graphics system

Cited by 83 publications

References 42 publications

Pannexin 1 mutation found in melanoma tumor reduces phosphorylation, glycosylation, and trafficking of the channel-forming protein

Pannexin 1 mutation found in melanoma tumor reduces phosphorylation, glycosylation, and trafficking of the channel-forming protein

SPOP mutation induces replication over-firing by impairing Geminin ubiquitination and triggers replication catastrophe upon ATR inhibition

The intracellular domains of the EphB6 and EphA10 receptor tyrosine pseudokinases function as dynamic signalling hubs

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