Publications by authors named "Jordan T B Stariha"

4 Publications

  • Page 1 of 1

Probing Protein-Membrane Interactions and Dynamics Using Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS).

Methods Mol Biol 2021 ;2263:465-485

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada.

Cellular membranes are a central hub for initiation and execution of many signaling processes. Integral to these processes being accomplished appropriately is the highly controlled recruitment and assembly of proteins at membrane surfaces. The study of the molecular mechanisms that mediate protein-membrane interactions can be facilitated by utilizing hydrogen-deuterium exchange mass spectrometry (HDX-MS). HDX-MS is a robust analytical technique that allows for the measurement of the exchange rate of backbone amide hydrogens with solvent to make inferences about protein structure and conformation. This chapter discusses the use of HDX-MS as a tool to study the conformational changes that occur within peripheral membrane proteins upon association with membrane. Particular reference will be made to the analysis of the protein kinase Akt and its activation upon binding phosphatidylinositol (3,4,5) tris-phosphate (PIP)-containing membranes to illustrate specific methodological principles.
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http://dx.doi.org/10.1007/978-1-0716-1197-5_22DOI Listing
June 2021

Defining How Oncogenic and Developmental Mutations of PIK3R1 Alter the Regulation of Class IA Phosphoinositide 3-Kinases.

Structure 2020 02 9;28(2):145-156.e5. Epub 2019 Dec 9.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8W 2Y2, Canada. Electronic address:

The class I phosphoinositide 3-kinases (PI3Ks) are key signaling 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 alter the activity of p110α/p110δ catalytic subunits. We find that the oncogenic Q572 truncation of PIK3R1 disrupts all p85-inhibitory inputs, with p110α being hyper-activated compared with 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 unique insight into isoform-specific regulation of p110s by p85α.
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http://dx.doi.org/10.1016/j.str.2019.11.013DOI Listing
February 2020

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

EMBO Rep 2020 02 12;21(2):e48441. Epub 2019 Dec 12.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, Canada.

The lipid kinase PI4KB, which generates phosphatidylinositol 4-phosphate (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 hydrogen-deuterium 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. Complex-disrupting 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 c10orf76-dependent 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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http://dx.doi.org/10.15252/embr.201948441DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7001497PMC
February 2020

Structural determinants of Rab11 activation by the guanine nucleotide exchange factor SH3BP5.

Nat Commun 2018 09 14;9(1):3772. Epub 2018 Sep 14.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC, V8W 2Y2, Canada.

The GTPase Rab11 plays key roles in receptor recycling, oogenesis, autophagosome formation, and ciliogenesis. However, investigating Rab11 regulation has been hindered by limited molecular detail describing activation by cognate guanine nucleotide exchange factors (GEFs). Here, we present the structure of Rab11 bound to the GEF SH3BP5, along with detailed characterization of Rab-GEF specificity. The structure of SH3BP5 shows a coiled-coil architecture that mediates exchange through a unique Rab-GEF interaction. Furthermore, it reveals a rearrangement of the switch I region of Rab11 compared with solved Rab-GEF structures, with a constrained conformation when bound to SH3BP5. Mutation of switch I provides insights into the molecular determinants that allow for Rab11 selectivity over evolutionarily similar Rab GTPases present on Rab11-positive organelles. Moreover, we show that GEF-deficient mutants of SH3BP5 show greatly decreased Rab11 activation in cellular assays of active Rab11. Overall, our results give molecular insight into Rab11 regulation, and how Rab-GEF specificity is achieved.
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http://dx.doi.org/10.1038/s41467-018-06196-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6138693PMC
September 2018