Publications by authors named "Noah J Harris"

3 Publications

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Biochemical Insight into Novel Rab-GEF Activity of the Mammalian TRAPPIII Complex.

J Mol Biol 2021 Jul 3;433(18):167145. Epub 2021 Jul 3.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8W 2Y2, Canada; Life Sciences Institute, Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada. Electronic address:

Transport Protein Particle complexes (TRAPP) are evolutionarily conserved regulators of membrane trafficking, with this mediated by their guanine nucleotide exchange factor (GEF) activity towards Rab GTPases. In metazoans evidence suggests that two different TRAPP complexes exist, TRAPPII and TRAPPIII. These two complexes share a common core of subunits, with complex specific subunits (TRAPPC9 and TRAPPC10 in TRAPPII and TRAPPC8, TRAPPC11, TRAPPC12, TRAPPC13 in TRAPPIII). TRAPPII and TRAPPIII have distinct specificity for GEF activity towards Rabs, with TRAPPIII acting on Rab1, and TRAPPII acting on Rab1 and Rab11. The molecular basis for how these complex specific subunits alter GEF activity towards Rab GTPases is unknown. Here we have used a combination of biochemical assays, hydrogen deuterium exchange mass spectrometry (HDX-MS) and electron microscopy to examine the regulation of TRAPPII and TRAPPIIII complexes in solution and on membranes. GEF assays revealed that TRAPPIII has GEF activity against Rab1 and Rab43, with no detectable activity against the other 18 Rabs tested. The TRAPPIII complex had significant differences in protein dynamics at the Rab binding site compared to TRAPPII, potentially indicating an important role of accessory subunits in altering the active site of TRAPP complexes. Both the TRAPPII and TRAPPIII complexes had enhanced GEF activity on lipid membranes, with HDX-MS revealing numerous conformational changes that accompany membrane association. HDX-MS also identified a membrane binding site in TRAPPC8. Collectively, our results provide insight into the functions of TRAPP complexes and how they can achieve Rab specificity.
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http://dx.doi.org/10.1016/j.jmb.2021.167145DOI Listing
July 2021

Disease-related mutations in PI3Kγ disrupt regulatory C-terminal dynamics and reveal a path to selective inhibitors.

Elife 2021 Mar 4;10. Epub 2021 Mar 4.

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

Class I Phosphoinositide 3-kinases (PI3Ks) are master regulators of cellular functions, with the class IB PI3K catalytic subunit (p110γ) playing key roles in immune signalling. p110γ is a key factor in inflammatory diseases and has been identified as a therapeutic target for cancers due to its immunomodulatory role. Using a combined biochemical/biophysical approach, we have revealed insight into regulation of kinase activity, specifically defining how immunodeficiency and oncogenic mutations of R1021 in the C-terminus can inactivate or activate enzyme activity. Screening of inhibitors using HDX-MS revealed that activation loop-binding inhibitors induce allosteric conformational changes that mimic those in the R1021C mutant. Structural analysis of advanced PI3K inhibitors in clinical development revealed novel binding pockets that can be exploited for further therapeutic development. Overall, this work provides unique insights into regulatory mechanisms that control PI3Kγ kinase activity and shows a framework for the design of PI3K isoform and mutant selective inhibitors.
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http://dx.doi.org/10.7554/eLife.64691DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955810PMC
March 2021

The substrate specificity of the human TRAPPII complex's Rab-guanine nucleotide exchange factor activity.

Commun Biol 2020 12 4;3(1):735. Epub 2020 Dec 4.

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

The TRAnsport Protein Particle (TRAPP) complexes act as Guanine nucleotide exchange factors (GEFs) for Rab GTPases, which are master regulators of membrane trafficking in eukaryotic cells. In metazoans, there are two large multi-protein TRAPP complexes: TRAPPII and TRAPPIII, with the TRAPPII complex able to activate both Rab1 and Rab11. Here we present detailed biochemical characterisation of Rab-GEF specificity of the human TRAPPII complex, and molecular insight into Rab binding. GEF assays of the TRAPPII complex against a panel of 20 different Rab GTPases revealed GEF activity on Rab43 and Rab19. Electron microscopy and chemical cross-linking revealed the architecture of mammalian TRAPPII. Hydrogen deuterium exchange MS showed that Rab1, Rab11 and Rab43 share a conserved binding interface. Clinical mutations in Rab11, and phosphomimics of Rab43, showed decreased TRAPPII GEF mediated exchange. Finally, we designed a Rab11 mutation that maintained TRAPPII-mediated GEF activity while decreasing activity of the Rab11-GEF SH3BP5, providing a tool to dissect Rab11 signalling. Overall, our results provide insight into the GTPase specificity of TRAPPII, and how clinical mutations disrupt this regulation.
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http://dx.doi.org/10.1038/s42003-020-01459-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7719173PMC
December 2020
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