Publications by authors named "Cameron J Powell"

7 Publications

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association with host mitochondria requires key mitochondrial protein import machinery.

Proc Natl Acad Sci U S A 2021 Mar;118(12)

Department of Biological Sciences, Dietrich School of Arts and Sciences, University of Pittsburgh, Pittsburgh, PA 15261;

Host mitochondrial association (HMA) is a well-known phenomenon during infection of the host cell. The locus () is required for HMA and encodes distinct paralogs of secreted dense granule effector proteins, some of which mediate the HMA phenotype (MAF1b paralogs drive HMA; MAF1a paralogs do not). To identify host proteins required for MAF1b-mediated HMA, we performed unbiased, label-free quantitative proteomics on host cells infected with type II parasites expressing MAF1b, MAF1a, and an HMA-incompetent MAF1b mutant. Across these samples, we identified ∼1,360 MAF1-interacting proteins, but only 13 that were significantly and uniquely enriched in MAF1b pull-downs. The gene products include multiple mitochondria-associated proteins, including those that traffic to the mitochondrial outer membrane. Based on follow-up endoribonuclease-prepared short interfering RNA (esiRNA) experiments targeting these candidate MAF1b-targeted host factors, we determined that the mitochondrial receptor protein TOM70 and mitochondria-specific chaperone HSPA9 were essential mediators of HMA. Additionally, the enrichment of TOM70 at the parasitophorous vacuole membrane interface suggests parasite-driven sequestration of TOM70 by the parasite. These results show that the interface between the vacuole and the host mitochondria is characterized by interactions between a single parasite effector and multiple target host proteins, some of which are critical for the HMA phenotype itself. The elucidation of the functional members of this complex will permit us to explain the link between HMA and changes in the biology of the host cell.
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http://dx.doi.org/10.1073/pnas.2013336118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7999873PMC
March 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 and mechanistic insights into the function of the unconventional class XIV myosin MyoA from .

Proc Natl Acad Sci U S A 2018 11 22;115(45):E10548-E10555. Epub 2018 Oct 22.

Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8P 5C2, Canada;

Parasites of the phylum Apicomplexa are responsible for significant morbidity and mortality on a global scale. Central to the virulence of these pathogens are the phylum-specific, unconventional class XIV myosins that power the essential processes of parasite motility and host cell invasion. Notably, class XIV myosins differ from human myosins in key functional regions, yet they are capable of fast movement along actin filaments with kinetics rivaling previously studied myosins. Toward establishing a detailed molecular mechanism of class XIV motility, we determined the 2.6-Å resolution crystal structure of the MyoA (TgMyoA) motor domain. Structural analysis reveals intriguing strategies for force transduction and chemomechanical coupling that rely on a divergent SH1/SH2 region, the class-defining "HYAG"-site polymorphism, and the actin-binding surface. In vitro motility assays and hydrogen-deuterium exchange coupled with MS further reveal the mechanistic underpinnings of phosphorylation-dependent modulation of TgMyoA motility whereby localized regions of increased stability and order correlate with enhanced motility. Analysis of solvent-accessible pockets reveals striking differences between apicomplexan class XIV and human myosins. Extending these analyses to high-confidence homology models of and MyoA motor domains supports the intriguing potential of designing class-specific, yet broadly active, apicomplexan myosin inhibitors. The successful expression of the functional TgMyoA complex combined with our crystal structure of the motor domain provides a strong foundation in support of detailed structure-function studies and enables the development of small-molecule inhibitors targeting these devastating global pathogens.
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http://dx.doi.org/10.1073/pnas.1811167115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6233092PMC
November 2018

A Toxoplasma gondii locus required for the direct manipulation of host mitochondria has maintained multiple ancestral functions.

Mol Microbiol 2018 06 11;108(5):519-535. Epub 2018 Apr 11.

Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA.

The Toxoplasma gondii locus mitochondrial association factor 1 (MAF1) encodes multiple paralogs, some of which mediate host mitochondrial association (HMA). Previous work showed that HMA was a trait that arose in T. gondii through neofunctionalization of an ancestral MAF1 ortholog. Structural analysis of HMA-competent and incompetent MAF1 paralogs (MAF1b and MAF1a, respectively) revealed that both paralogs harbor an ADP ribose binding macro-domain, with comparatively low (micromolar) affinity for ADP ribose. Replacing the 16 C-terminal residues of MAF1b with those of MAF1a abrogated HMA, and we also show that only three residues in the C-terminal helix are required for MAF1-mediated HMA. Importantly these same three residues are also required for the in vivo growth advantage conferred by MAF1b, providing a definitive link between in vivo proliferation and manipulation of host mitochondria. Co-immunoprecipitation assays reveal that the ability to interact with the mitochondrial MICOS complex is shared by HMA-competent and incompetent MAF1 paralogs and mutants. The weak ADPr coordination and ability to interact with the MICOS complex shared between divergent paralogs may represent modular ancestral functions for this tandemly expanded and diversified T. gondii locus.
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http://dx.doi.org/10.1111/mmi.13947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245547PMC
June 2018

Dissecting the molecular assembly of the MyoA motility complex.

J Biol Chem 2017 11 25;292(47):19469-19477. Epub 2017 Sep 25.

From the Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia V8P 5C2, Canada and

Apicomplexan parasites such as rely on a unique form of locomotion known as gliding motility. Generating the mechanical forces to support motility are divergent class XIV myosins (MyoA) coordinated by accessory proteins known as light chains. Although the importance of the MyoA-light chain complex is well-established, the detailed mechanisms governing its assembly and regulation are relatively unknown. To establish a molecular blueprint of this dynamic complex, we first mapped the adjacent binding sites of light chains MLC1 and ELC1 on the MyoA neck (residues 775-818) using a combination of hydrogen-deuterium exchange mass spectrometry and isothermal titration calorimetry. We then determined the 1.85 Å resolution crystal structure of MLC1 in complex with its cognate MyoA peptide. Structural analysis revealed a bilobed architecture with MLC1 clamping tightly around the helical MyoA peptide, consistent with the stable 10 nm measured by isothermal titration calorimetry. We next showed that coordination of calcium by an EF-hand in ELC1 and prebinding of MLC1 to the MyoA neck enhanced the affinity of ELC1 for the MyoA neck 7- and 8-fold, respectively. When combined, these factors enhanced ELC1 binding 49-fold (to a of 12 nm). Using the full-length MyoA motor (residues 1-831), we then showed that, in addition to coordinating the neck region, ELC1 appears to engage the MyoA converter subdomain, which couples the motor domain to the neck. These data support an assembly model where staged binding events cooperate to yield high-affinity complexes that are able to maximize force transduction.
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http://dx.doi.org/10.1074/jbc.M117.809632DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5702683PMC
November 2017

The structure of Plasmodium falciparum 3D7_0606800 reveals a bi-lobed architecture that supports re-annotation as a Venus Flytrap protein.

Protein Sci 2017 Sep 26;26(9):1878-1885. Epub 2017 Jul 26.

Biochemistry & Microbiology, University of Victoria, Victoria, British Columbia, V8W 3P6, Canada.

Plasmodium falciparum, the causative agent of malaria, employs a diverse array of surface displayed proteins to promote dissemination and establish infection in the human host. Of these, Pf3D7_0606800 is highly immunogenic and has been designated a potential top 10 candidate for inclusion in a multicomponent malarial vaccine. The role of Pf3D7_0606800 in parasite biology, however, is unknown and its characterization has been complicated by a lack of sequence identity with proteins of known structure or function. Towards elucidating Pf3D7_0606800 function, we determined its structure to a resolution of 2.35 Å using selenium single wavelength anomalous dispersion. A bi-lobed architecture displays the core structural hallmarks of Venus Flytrap (VFT) proteins prompting us to re-annotate Pf3D7_0606800 as PfVFT1. Structural analysis further revealed an extended inter-lobe groove that, when interrogated by molecular docking, appears well suited to bind peptide-based ligands. Collectively, our structural characterization of the highly antigenic P. falciparum surface protein PfVFT1 provides intriguing functional insight and establishes a structural template that could prove valuable for malaria vaccine engineering studies.
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http://dx.doi.org/10.1002/pro.3218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5563136PMC
September 2017
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