PhD Exit Seminars: Andrew Alexander and Franco Li

Nicolas Coutin

“Method Development for High Throughput Biology,” by Nicolas Coutin, doctoral candidate, Corey Nislow Lab, UBC

Abstract:

Living things are made from thousands of individual, interacting pieces. To understand these systems, we will need to study many or all the pieces at once. To study biology this way, we will need tools that consider the whole system and how its pieces interact. This work describes methods we developed to study the many components of biological systems at once. It presents a technique that discovers interactions between the molecular machines that act on the genome. This work also demonstrates an application that lets its users analyze thousands of individual experiments interactively.

John Young

“Exploring the interactome of the bacterial Sec translocon,” by John Young, doctoral candidate, Franck Duong Lab, UBC.

Abstract:

Many periplasmic and extracellular Escherichia coli (E. coli) proteins are transported across the inner bacterial membrane through the highly conserved heterotrimeric SecYEG protein-conducting channel. During post-translational translocation, polypeptide substrates are driven across the membrane through SecYEG by the ATPase SecA, which binds to SecYEG and couples nucleotide hydrolysis to polypeptide movement. In the first part of this thesis, we study the dynamics of SecYEG-SecA interactions. We show that SecA is a highly dynamic enzyme, repeatedly binding to and dissociating from SecYEG during substrate translocation. Using two model Sec-dependent protein substrates, we show that the importance of these dynamics for efficient translocation depends on the length of the translocating protein substrate. In the second part of this thesis, we turn to quantitative proteomics to identify novel interactors of the SecYEG complex. Previous studies have identified and validated a series of membrane embedded interactors of SecYEG using classical detergent-based methods. However, it is possible that other important interactors of the Sec translocon may exist which have not yet been identified by detergent-based proteomic methods – the difficulties of using detergent-based methods to identify and characterize transient interactors of membrane proteins and complexes are well-documented. Here, we employ the peptidisc – a “one-size-fits-all” membrane mimetic – to identify and characterize potentially novel interactors of the Sec translocon in detergent-free conditions. One of the most notable interactions identified in this work is a super-complex between the Sec translocon and the outer membrane embedded Bam complex, which is required for insertion of outer membrane proteins (OMPs). This observation is particularly astonishing and has implications for our understanding of outer membrane protein biogenesis. Finally, we develop a functionalized variant of the peptidisc scaffold and demonstrate its utility for isolation of the membrane proteome.  As a simple case study, we employ the functionalized peptidisc scaffold to survey changes in the membrane proteome caused by altered gene expression. Potential future applications of the peptidisc membrane mimetic in the fields of membrane protein biochemistry and membrane proteomics will also be discussed.

Monday, March 23, 2020 at 2:30 pm, LSC #3

“Tumor cells highjack diverse cellular  processes to maintain redox balance”, by Poul Sorensen, Professor, Department of Pathology & Laboratory Medicine, UBC, Distinguished Scientist, BC Cancer Research Centre Johal Chair in Childhood Cancer Research, UBC.

In aggressive sarcomas such as Ewing sarcoma (EwS) and osteosarcoma (OS), the single-most powerful predictor of poor outcome is metastatic disease, highlighting the critical need to identify new factors driving metastasis in these diseases. Metastasis is widely regarded as a very inefficient process, likely due to diverse stress forms that can potentially cull pre-metastatic cancer cells during the metastatic cascade, including hypoxia in primary tumors, anoikis stress in the circulation, and increased oxidative stress at distant organs prior to colonization. Adaptation to such conditions requires rapid stress-alleviating plasticity to confer fitness for metastatic progression, but mechanisms remain elusive. Studies in our laboratory indicate that mitigation of oxidative stress, not only during local invasion at the primary tumor site, but also in the circulation during dissemination, and potentially as part of colonization at distant sites, is critical for childhood sarcoma metastatic capacity. Indeed, EwS and OS cells appear to utilize many different strategies to maintain redox balance, such as the induction of anti-oxidant pathways involving NRF2, or through other mechanisms such as alterations in amino acid transporter systems to facilitate production of glutathione and other anti-oxidants via amino acid metabolism. Some of these pathways are transcriptionally regulated, such as through direct activation by oncogenic transcription factors, while others are regulated through rapid translational activation by pioneer translation factors such as YB-1. Examples of each of these processes will be discussed.

Monday, March 9, 2020 at 2:30 pm, LSC#3

Host: Dr. Eric Jan