Matthew Solomonson - Doctoral Exam

“Characterization of Cricket Paralysis Virus Virus-Host Interaction and Viral Protein Synthesis,”
by Anthony Khong, Doctoral Student in Eric Jan Lab.

Tuesday, July 28, 2015 9:00 AM – Room 1410, Life Sciences Centre, 2350 Health Sciences Mall

Thesis title, “Structure, proteolysis, and evolution of secreted tuberculosis virulence factors,” Matthew Solomonson.

Monday, July 27, 2015 at 1:00 pm, Rm 5310, Life Sciences Centre, 2350 Health Science Mall

ABSTRACT
Mycobacterium tuberculosis (TB) uses the ESX-1 type VII secretion system to export proteins to its cell surface, which permeabilize the host macrophage phagosomal membrane, allowing the bacterium to escape and spread to new cells. The structure of the type VII membrane complex and how it mediates this function is unknown, but it is hypothesized that some of the secreted proteins form an extracellular appendage that facilitates membrane lysis or direct secretion of virulence factors into the host cytoplasm. This thesis investigates the structural relationship between one of these secreted proteins, EspB, and a protease that processes it, MycP1. The x-ray crystallographic structures of both proteins are determined. EspB is shown to form a multimer with heptameric stoichiometry, and an EM reconstruction of this multimer is generated and used to create a model of the oligomer using symmetric Rosetta docking. The final model is supported by mass spectrometry-based detection of chemically cross-linked peptides from adjacent subunits. We use mass spectrometry to determine how EspB is proteolytically processed during secretion and discuss the effect of this processing event on the EspB ultrastructure. Finally, the structure of one of the membrane apparatus proteins, EccB1 is determined, revealing structural homology to a phage lysin. The combination of x-ray crystallography, EM, modeling, and mass-spectrometry provides an exciting first glimpse at the structure and function of the type VII secretion system – a critical factor in the TB pathogenesis cycle.

Thesis title, “Structural and Functional Characterization of Components of Bacterial Type III Secretion Systems,” by Brianne Burkinshaw.

Monday, July 20, 2015 at 9:00 am, Rm 203, Graduate Student Centre, 6371 Crescent Road.

ABSTRACT
Many Gram-negative pathogens use a type III secretion system (T3SS) to
inject effector proteins into the host cytoplasm, where they manipulate host processes
to the advantage of the bacterium. The T3SS is composed of a cytoplasmic export
apparatus, a membrane-spanning basal body with a central channel formed by the
inner rod, an extra-cellular needle filament and a translocon complex that inserts in
the host membrane. In this thesis, proteins involved in T3SS assembly, as well as a
T3SS effector protein were structurally and functionally characterized. The structure
of EtgA, a T3SS-associated peptidoglycan (PG)-cleaving enzyme from EPEC was
solved. The EtgA active site has features in common with lytic transglycosylases
(LTs) and hen egg-white lysozyme (HEWL). EtgA contains an aspartate that aligns
with lysozyme Asp52 (a residue critical for catalysis), a conservation not observed in
LT families to which the conserved T3SS enzymes were presumed to belong.
Mutation of the EtgA catalytic glutamate conserved across LTs and HEWL, and this
differentiating aspartate diminishes type III secretion in vivo, supporting its essential
role in T3SS assembly. EtgA forms a complex with the T3SS inner rod component,
which enhances PG-lytic activity of EtgA in vitro, providing localization and regulation
of the lytic activity to prevent cell lysis. After assembly of the basal body and needle,
the gatekeeper protein ensures the translocon assembles at the needle tip prior to
secretion of effector proteins. The gatekeeper from EPEC (SepL) was crystallized and
it was shown that it has three X-bundle domains, which likely mediate protein-protein
interactions to control translocon and effector secretion. Through comparison of
SepL to structurally characterized homologs, revealed a number of conserved
residues, which may be required to regulate secretion of translocators or effectors.
Finally, SopB, a Salmonella effector protein, in complex with host Cdc42, an essential
Rho GTPase that regulates critical events in eukaryotic cytoskeleton organization and
membrane trafficking was structurally characterized. Structural and biochemical
analysis of the SopB/ Cdc42 complex shows that SopB structurally and functionally
mimics a host guanine nucleotide dissociation inhibitor (GDI) by contacting key
residues in the regulatory switch regions of Cdc42 and slowing Cdc42 nucleotide
exchange.

Thesis title, “Insights into Cargo Adaptor Function Through The Study of Novel Interactors” by Bjorn Bean.

Wednesday, July 8, 2015 at 9:00 am in Room 200, Graduate Student Centre, 6371 Crescent Road

ABSTRACT
In Eukaryotes, luminal and transmembrane proteins are moved to their
functional locations by conserved membrane trafficking machinery. In this process,
cargo adaptors bind motifs present on cargo, indirectly linking the proteins to coats,
which deform membranes and form transport vesicles. Here, cargo adaptor
recruitment and cargo recognition was studied by characterizing associated factors in
the budding yeast Saccharomyces cerevisiae. Possible cargo adaptor-associated
factors were identified in a proteomics study that grouped protein-protein interactions
into 501 putative membrane associated complexes using a Markov clustering
algorithm. Two clusters were selected for this work.
The first contained the uncharacterized protein Ssp120 with the endoplasmic
reticulum-to-Golgi trafficking complex Emp46/Emp47. Ssp120 stably interacted with
the Emp46/Emp47 complex and depended on Emp47 for its punctate localization.
The C-terminus of Ssp120 mediated the interaction. Homology with human MCFD2
suggests that Ssp120 may link a subset of cargo to Emp46/Emp47.
The second cluster was comprised of retromer, an endosome-to-Golgi
trafficking complex, and the Rab5-family guanine nucleotide exchange factor (GEF)
Muk1. Both Muk1 and the other known Rab5-family GEF, Vps9, interacted with
retromer and the presence of at least one was required for retromer recruitment to
endosomes. Additionally, a new VPS9 domain-containing protein present was
identified and shown to complement loss of MUK1 and VPS9. Retromer recruitment
was shown to be dependent on putative GEF catalytic residues and the presence of
their target Rabs. Furthermore, loss of GEFs resulted in mislocalization of the
potential Rab5 effector, Vps34, and its lipid product, phosphatidylinositol 3-phosphate
(PI3P), to the vacuolar membrane. As retromer is recruited by PI3P, the data support
a positive feedback model whereby retromer interacts with GEFs to indirectly modify
the lipid composition of the membrane allowing further localized recruitment.
This study validates the approach of studying novel interactors of cargo
recognition complexes to better understand their function. It suggests that Ssp120
may recognize a subset of Emp46-Emp47 cargo, indicating that an associated factor
can diversify the proteins recognized by a given cargo adaptor. Furthermore, the work
on retromer suggests a novel mechanism for the reinforcement of cargo selective
complex recruitment that may be conserved in humans.

Week #4, Friday, June 19 from noon-1:00 pm in LSC 3.

Dr. Patrick Hau Wing Chan,  Postdoctoral Fellow in Thibault Mayor Lab presents,

“Proteomic profiling of the [PSI+] yeast prion strain by quantitative mass spectrometry”

Prions proteins can adopt a second conformation that induces the formation of amyloid fibrils that are responsible for the transmissible spongiform encephalopathy in mammals. In contrast to mammalian prion, yeast prion proteins are controversially considered as advantageous for natural survival rather than being toxic. [PSI+] describes the yeast prion state in which the translational termination factor, Sup35, forms amyloid fibrils, resulting in an increase of non-sense suppression and giving a distinct phenotype. Due to non-sense suppression in [PSI+], part of its proteome might have an increased amount of C-terminal extended sequences, which could potentially affect the stability of the proteins positively or negatively. By differentiating the proteomes between [psi-] and [PSI+] states and identifying the C-terminal extended proteins, it would provide us some insights for what biological pathways are affected and how they provide survival advantages for being in prion state.