Probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium exchange–MS (HDX–MS)

Vadas, Oscar; Burke, John E.

doi:10.1042/bst20150065

Cited by 57 publications

(46 citation statements)

References 98 publications

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“…HDX-MS is thus a potent tool to determine proteinprotein, protein-ligand, and protein-membrane interactions [37][38][39]. HDX-MS is an analytical technique that measures the exchange rate of amide hydrogens in proteins.…”

Section: Resultsmentioning

confidence: 99%

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

et al. 2019

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The lipid kinase PI4KB, which generates phosphatidylinositol 4phosphate (PI4P), is a key enzyme in regulating membrane transport and is also hijacked by multiple picornaviruses to mediate viral replication. PI4KB can interact with multiple protein binding partners, which are differentially manipulated by picornaviruses to facilitate replication. The protein c10orf76 is a PI4KB-associated protein that increases PI4P levels at the Golgi and is essential for the viral replication of specific enteroviruses. We used hydrogendeuterium exchange mass spectrometry to characterize the c10orf76-PI4KB complex and reveal that binding is mediated by the kinase linker of PI4KB, with formation of the heterodimeric complex modulated by PKA-dependent phosphorylation. Complexdisrupting mutations demonstrate that PI4KB is required for membrane recruitment of c10orf76 to the Golgi, and that an intact c10orf76-PI4KB complex is required for the replication of c10orf76dependent enteroviruses. Intriguingly, c10orf76 also contributed to proper Arf1 activation at the Golgi, providing a putative mechanism for the c10orf76-dependent increase in PI4P levels at the Golgi.

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Section: Resultsmentioning

confidence: 99%

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

et al. 2019

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“…To investigate the molecular mechanisms that lead to the hyper-activation of the p85α-Q572* truncation mutant when in complex with p110α, we used HDX-MS to probe for activating conformational changes between the WT and the p85α-Q572* truncation. HDX-MS is a powerful analytical technique that measures the exchange rate of amide hydrogens in solution, and has been extensively used to define the conformational mechanisms that underlie PI3K activation by both activating stimuli and disease linked mutations (Burke, 2019;Masson et al, 2017;Vadas and Burke, 2015). Essential to this technique is the generation of peptic peptides spanning the entire sequence to allow for the localisation of deuterium incorporation.…”

Section: The P85α Oncogenic Q572* Truncation Of the Ish2 Coiled-coil mentioning

confidence: 99%

Defining how oncogenic and developmental mutations ofPIK3R1alter the regulation of class IA phosphoinositide 3-kinases

Dornan

Stariha

Rathinaswamy

et al. 2019

Preprint

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The class IA PI3Ks are key signalling enzymes composed of a heterodimer of a p110 catalytic subunit and a p85 regulatory subunit, with PI3K mutations being causative of multiple human diseases including cancer, primary immunodeficiencies, and developmental disorders.Mutations in the p85α regulatory subunit encoded by PIK3R1 can both activate PI3K through oncogenic truncations in the iSH2 domain, or inhibit PI3K through developmental disorder mutations in the cSH2 domain. Using a combined biochemical and hydrogen deuterium exchange mass spectrometry approach we have defined the molecular basis for how these mutations activate both the p110α/p110δ catalytic subunits. We find that the oncogenic Q572* truncation of PIK3R1 disrupts all inhibitory inputs, with p110α being hyper-activated compared to p110δ. In addition, we find that the R649W mutation in the cSH2 of PIK3R1 decreases sensitivity to activation by receptor tyrosine kinases. This work reveals novel insight into isoform specific regulation of p110s by p85α.Keywords: PI3K, p110, p85, phosphoinositide 3-kinase, PI3K-Akt, HDX-MS, hydrogen exchange, phosphoinositides, PIK3CA, PIK3R1Defining the p85 mechanisms that mediate the inhibition and activation of PI3K Highlights-An oncogenic variant of p85α, Q572*, leads to hyper-activation of p110α compared to p110δ -HDX-MS revealed that Q572* leads to disruption of all inhibitory interfaces in p110α -A SHORT syndrome mutation in p85α leads to decreased sensitivity to RTKs for p110α/δ Defining the p85 mechanisms that mediate the inhibition and activation of PI3K

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“…Protection from amide exchange is mediated by involvement in secondary structure, and measuring exchange rates allows determination of protein conformational dynamics. This technique has been deployed to probe the interaction of lipid modifying enzymes with membranes and membrane proteins [28][29][30][31][32]. SK1 was incubated in the presence and absence of membrane vesicles composed of 40% PC, 35% PE, 20% PA, and 5% cholesterol.…”

Section: Mapping Of Sk1/liposome Interface With Hydrogen/deuterium Exmentioning

confidence: 99%

An intrinsic lipid-binding interface controls sphingosine kinase 1 function

Pulkoski-Gross

Jenkins

Truman

et al. 2018

Journal of Lipid Research

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Sphingosine kinase 1 (SK1) is required for production of sphingosine-1-phosphate (S1P) and thereby regulates many cellular processes-including cellular growth, immune cell trafficking, and inflammation. To produce S1P, SK1 must access sphingosine directly from membranes. However, the molecular mechanisms underlying SK1's direct membrane interactions remain unclear. We used hydrogen-deuterium exchange mass spectrometry to study interactions of SK1 with membrane vesicles. Using the CRISPR/Cas9 technique to generate HCT116 cells lacking SK1, we explored the effects of membrane interface disruption and the function of the SK1 interaction site. Disrupting the interface resulted in reduced membrane association and decreased cellular SK1 activity. Moreover, SK1-dependent signaling, including cell invasion and endocytosis, was abolished upon mutation of the membrane-binding interface. Of note, we identified a positively charged motif on SK1 that is responsible for electrostatic interactions with membranes. Furthermore, we demonstrated that SK1 uses a single contiguous interface, consisting of an electrostatic site and a hydrophobic site, to interact with membrane-associated anionic phospholipids. Altogether, these results define a composite domain in SK1 that regulates its intrinsic ability to bind membranes and indicate that this binding is critical for proper SK1 function. This work will allow for a new line of thinking for targeting SK1 in disease.

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Probing the dynamic regulation of peripheral membrane proteins using hydrogen deuterium exchange–MS (HDX–MS)

Cited by 57 publications

References 98 publications

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

Characterization of the c10orf76‐PI4KB complex and its necessity for Golgi PI4P levels and enterovirus replication

Defining how oncogenic and developmental mutations ofPIK3R1alter the regulation of class IA phosphoinositide 3-kinases

An intrinsic lipid-binding interface controls sphingosine kinase 1 function

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