Project Code: BCH 1S RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2017‐2018 SUMMER Name and Title: John Glover (Associate Professor) Department: Biochemistry TITLE OF RESEARCH PROJECT: The Interaction of Hsp104 with a Yeast Prion NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: Background: Hsp104 is a molecular chaperone in yeast that specializes in the extraction of proteins trapped in aggregates. Hsp104 is fundamentally important for the survival of yeast exposed to protein unfolding stresses like extreme heat. Hsp104 also is important for the propagation of self‐replicating protein aggregates. These aggregates are transmitted through cytosolic transfer from mother to daughter cells during budding and thereby serve as “protein‐only” genetic elements. This process is parallel to the “protein‐only” the transmission of mammalian prion disease from afflicted animals to healthy animals. Hsp104 is required for prion propagation in yeast. Objective: We have mapped the physical interaction between Hsp104 and the yeast prion protein Sup35. Our objective is to determine how this peptide interacts directly with Hsp104 Methodology: The short 20‐amino acid region that interacts directly with Hsp104 will serve as a probe to determine how this binding takes place. Our evidence (unpublished) and that of others suggests that this binding is dependent on the N‐terminal domain of Hsp104, which is an independently‐folded domain of approximately 150 amino acids. We will use immobilized peptide to monitor binding of Hsp104 full‐length protein and N‐terminal, and surface plasmon resonance (SPR) to measure binding affinities. Time permitting, once we have fully characterized the binding interaction by biochemical means we will explore cross‐linking or NMR methodologies (depending on the size of the minimal Hsp104 binding segment) to determine where on Hsp104 the prion‐derived peptide binds. The student will be trained in a broad range of biochemical and molecular biological techniques including simple cloning, DNA preparation and analysis, peptide synthesis, protein expression and purification, chemical coupling of peptides to solid phase supports, and analysis of binding through SDS‐PAGE analysis of fractions or by analysis of SPR binding curves. Suggested reading: Winkler et al., Chaperone networks in protein disaggregation and prion propagation. J Struct Biology 179: 2152–
160 (2012) Helsen & Glover, Insight into Molecular Basis of Curing of [PSI+] Prion by Overexpression of 104‐kDa Heat Shock Protein (Hsp104) J. Biol Chem 287:542–556 (2012) Helsen & Glover, A new perspective on Hsp104‐mediated propagation and curing of the yeast prion [PSI+]. Prion. 6:234‐9 (2012) DESCRIPTION OF STUDENT PARTICIPATION: The student will be expected to read appropriate background literature with guidance from the supervisor. Experimental planning and training in various techniques will be provided by the supervisor, with the student carrying out procedures on a semi‐independent basis. As the project progresses the student will participate to a greater extent in planning experiments. The student will be expected to keep accurate and complete records of their experimental work and the notebook will be viewed regularly by the supervisor who will make suggestions for improvement of record keeping. The student will be expected to meet regularly with the supervisor and attend weekly lab meetings to discuss work in progress and review current literature pertinent to the work being carried out MARKING SCHEME (assignments with weight and due date): 1. Project Proposal (2 pages plus figures and references, due within 3‐4 weeks after starting) 10% 2. Oral Project Presentation (20 min within 1 month of starting) 10% 3. Lab performance, work ethic, lab notes, participation, etc. 30% with 10% assigned midterm 4. Final Oral Presentation (20 min plus ability to answer questions) 20% 5. Final Lab Report (10 pages plus figures and references) 30% Project Code: BCH 2S RESEARCH OPPORTUNITY PROGRAM 299Y/399Y PROJECT DESCRIPTIONS 2017‐2018 SUMMER Name and Title: Walid A. Houry, Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: The Development of Novel Antibiotics NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: In recent years, there has been an alarming trend of increased bacterial infections caused by strains resistant to most known drugs. As a result, diseases that were thought to be controlled by currently available antibiotics are re‐emerging not only in developing countries but also in industrialized nations, especially in clinical settings such as hospitals. Therefore, there is an urgent need for the development of new types of antibiotics that can be used to effectively treat multidrug resistant bacteria. In this project, we propose to screen and develop a novel class of antibacterial drugs that can activate highly‐conserved, tightly‐regulated, self‐compartmentalizing cylindrical proteases in bacterial cells. One such protease is ClpP. On its own, ClpP can only degrade small peptides and not folded proteins. The binding of unfoldase chaperones to ClpP is required for the degradation of native proteins. ClpP has recently been validated as a novel molecular target for antibacterial drug development. We aim to develop and identify novel compounds that allow ClpP and other such cylindrical proteases to indiscriminately degrade folded proteins eventually causing bacterial cell death. The efficacy of the compounds will be tested using model infectious bacterial systems. These compounds will define a new class of antibiotics, namely activators of self‐compartmentalizing proteases, which we will refer to as ACPs. Students in BCH299 will work on characterizing the biochemical consequences of the binding of ACPs to the cylindrical proteases using purified proteins and pertinent biophysical methods. Students will not be involved in working with any pathogenic bacteria. DESCRIPTION OF STUDENT PARTICIPATION: The student will be involved in cloning different genes using standard molecular biology techniques, in purifying proteins mainly using His‐tag/Ni‐NTA, and in carrying out ATPase and binding assays using size exclusion chromatography with purified proteins. Students will work closely with a senior graduate student or postdoctoral fellow. MARKING SCHEME (assignments with weight and due date): 5% two‐page report due last week in mid‐June 10% 20‐minute presentation in the last week in mid‐June 25% 15‐page report at end of term in August 15% 30‐min presentation at end of term in August 45% work in the laboratory Project Code: BCH 3S RESEARCH OPPORTUNITY PROGRAM 299Y/399Y PROJECT DESCRIPTIONS 2017‐2018 SUMMER Name and Title: Dr. Warren Lee, Assistant Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: Mechanisms and Manipulation of Endothelial Permeability during Inflammation NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: Every blood vessel in the body is lined with a specialized layer of polarized cells known as endothelium. An essential function of the endothelial monolayer is the regulation of barrier integrity, which prevents the leakage of plasma and proteins out of the circulation while still permitting the flux of nutrients and immune cells to target tissues. In principle, permeability of the endothelial monolayer can reflect contributions from leaking between endothelial cells (paracellular leak) and through individual endothelial cells (transcellular leak, or transcytosis). It is widely accepted that paracellular leak predominates during inflammatory states such as sepsis and acute lung injury. We study paracellular leak during inflammation; for instance, using the influenza A virus as a model pathogen, we investigate how the virus induces lung endothelial permeability to cause pulmonary edema, a characteristic clinical feature of severe influenza infections in humans. We have reported effects of the virus on lung endothelial viability and on tight junction integrity; interestingly, at least some of the effect of the virus on endothelial barrier integrity is independent of viral replication and involves degradation of the tight junction constituent claudin‐5. Remarkably, there are no treatments for microvascular leak so identifying and testing potential endothelial barrier‐enhancing compounds is a major area of interest for my lab. The methodology used includes high‐resolution live cell imaging as well as traditional biochemical and molecular biology techniques and some mouse work. For further details, please consult the lab website (warrenleelab.com) DESCRIPTION OF STUDENT PARTICIPATION: Enthusiasm and industriousness are essential. Under appropriate supervision, the student will perform primary cell culture, live cell imaging, immunoblotting and immunofluorescence and mouse work. The student will be taught analytical and presentation skills. MARKING SCHEME (assignments with weight and due date): 2‐page Interim report 25%, due August 1st Lab journal 35% ‐ due at end of term Attendance and participation in lab meetings, journal club 15% Final oral presentation at lab meeting on the project 25% ‐ at end of term Project Code: BCH 4S RESEARCH OPPORTUNITY PROGRAM 299Y/399Y PROJECT DESCRIPTIONS 2017‐2018 SUMMER Name and Title: Trevor Moraes Associate Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: Structural and Functional Examination of Membrane Protein Interaction Surfaces NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: Disease‐causing bacteria rely on the acquisition of a diverse set of nutrients from their host environment to engage in successful pathogenesis. Recent studies have shown that host and microbiota‐derived carbohydrates play critical roles in regulating the behavior pathogens such as enterohemorrhagic Escherichia coli (EHEC) O157:H7, Salmonella typhimurium and Clostridium difficile. In order to acquire these nutrients, these Gram‐
negative pathogens are reliant on specific transport machineries termed binding protein‐
dependent transporters (BPDTs) to transport solutes such as amino acids, sugars and metal ions across their membranes (Sit el al. PLoS Pathogens 2015). The goal of this research is to analyze the interactions between these proteins and the nutrients that they steal from their hosts. The project will involve purifying proteins and examining their interactions using biophysical methods including; X‐ray protein crystallography to determine the 3D structure of proteins; biolayer interferometry (BLI), or microscale thermophoresis (MST) that can be used to measure the affinities between proteins and small molecules; finally the students will perform bacterial growth assays with difference versions of the transporters to test how mutations affect bacterial fitness and the ability for the bacteria to infect a host. DESCRIPTION OF STUDENT PARTICIPATION: Students will be paired with a research technician and a graduate student who will train them on each aspect of the project including creating site directed mutants of proteins involved in Ion translocation and then expressing, purifying and characterizing these mutants. The students will participate in the cloning (PCR amplifications, ligations and transformations) and expression of the derivative proteins. Utilizing affinity, ion exchange and size exclusion chromatography on an FPLC the students will purify these proteins. Purified protein will be analyzed to determine binding affinities compared to the wild‐type protein (methods include: BLI, MST). Structures of interesting mutant proteins will be determined using protein crystallography. A dedicated weekly meeting timeslot between the ROP student, research technician, graduate student and myself will be assigned and used to monitor progress and discuss the upcoming research plan. Our new location in MaRS, has study carrel space directly opposite the PIs office so there will also be plenty of casual discussions. MARKING SCHEME (assignments with weight and due date): Students will be evaluated on presentations and participation in lab meetings, lab notebook and lab performance (40% ‐lab members will help in the evaluation), a research proposal (20%, 1pg “summary page” ~ due 3‐4 weeks after starting the project followed by a 5pg NSERC style research proposal @ 4‐8 weeks), a midterm report on results (15%‐ detailed methods and results due midway through term), and an end of term report (25% due on the last day of classes – Journal of Biological Chemistry style report). Project Code: BCH 5S RESEARCH OPPORTUNITY PROGRAM 299Y/399Y PROJECT DESCRIPTIONS 2017‐2018 SUMMER Name and Title: Dr. Haley Wyatt, Assistant Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: Regulation of the Human SLX1 Nuclease by Homodimerisation NUMBER OF STUDENT PLACES AVAILABLE: 4 OBJECTIVES AND METHODOLOGY: Background Human cells contain sophisticated networks to repair DNA damage. These pathways safeguard genome integrity, ensure proper cell function and help protect us from developing diseases like cancer. Most DNA repair pathways rely on ‘molecular scissors’ called structure‐selective endonucleases (SSEs) to remove toxic DNA structures that form during DNA repair (and normal cellular growth). In human cells, a SSE called SLX1‐SLX4 is critical for DNA repair and genome stability. The biological importance of SLX1‐SLX4 is underscored by the fact that mutations in SLX4 are associated with Fanconi anemia (FA), a rare genetic disease characterized by genome instability and cancer predisposition. My lab aims to understand the biological and biochemical mechanisms that regulate the nuclease activity of SLX1‐SLX4. We have previously shown that Candida glabrata Slx1 is a stable homodimer in the absence of Slx4, an architecture that renders the enzyme catalytically inactive (Gaur et al., 2015). The presence of Slx4 triggers the displacement of the homodimer, leading to the formation of a catalytically active Slx1‐Slx4 heterodimer. This led us to propose a novel mechanism of Slx1 regulation ‐ inhibitory homodimerization. Our current studies are focused on exploring the biochemical and biological significance of this regulatory mechanism in human cells. This information will provide critical insight into exactly how SLX1‐SLX4 operates and executes accurate DNA repair. Our long‐term goal is to harness this information and develop strategies to alleviate the defects observed in FA patients harboring mutations in the SLX4 gene. Objectives The goals of this research project are three‐fold: 1. Determine if the nuclease activity of human SLX1 is negatively regulated by homodimerisation 2. Engineer a constitutively‐monomeric SLX1 mutant (called SLX1ONE) 3. Assess the phenotypes of human cells expressing SLX1ONE Methodology The formation and stability of human SLX1 homodimers will be investigated in vitro using recombinant proteins, purified from bacteria, and biophysical methods, including analytical ultracentrifucation (AUC) and multi‐angle light scattering (MALS) in line with size‐exclusion chromatography (SEC). In parallel, SLX1 fusion proteins containing unique affinity tags will be cloned, expressed in human cells, and tested for their ability to interact using co‐immunoprecipitation and western blotting. The functional relevance of SLX1 homodimerisation will be ascertained by cloning a dimerization‐defective mutant (SLX1ONE) and assessing its nuclease activity in vitro and in human cells. Specifically, SLX1ONE will be purified from bacteria and tested for DNA nuclease activity in vitro. In a related approach, SLX1ONE will be expressed in human cells that lack endogenous SLX1. By assessing the DNA repair phenotypes of the mutant cells, the student(s) will provide novel insight into the biological significance of SLX1. Suggested Reading 1. Gaur et al., Structural and mechanistic analysis of the Slx1‐Slx4 endonuclease. Cell Rep 10(9):1467‐76 (2015). 2. Wyatt et al., Coordinated actions of SLX1‐SLX4 and MUS81‐EME1 for Holliday junction resolution in human cells. Mol Cell 52(2):234‐47 (2013). DESCRIPTION OF STUDENT PARTICIPATION: This project can be tailored to the strengths and interests of individual students and is ideally suited for collaboration between students. The student(s) will be directly supervised by Dr. Wyatt and a research technician. With guidance from Dr. Wyatt, the student(s) will be expected to read appropriate background literature. Dr. Wyatt and a research technician will provide experimental planning and training. The student(s) will be expected to conduct experiments on a semi‐independent basis, with the expectation that they will become more independent as the project progresses. Each student will be responsible for keeping accurate and complete records of their experimental lab work. Dr. Wyatt will review these records on a weekly basis. Dr. Wyatt will also provide weekly feedback on each student’s progress. The student(s) will have ample opportunity to learn and/or enhance their communication skills through weekly meetings with Dr. Wyatt, participation in lab meetings, and interactions with neighboring researchers. The student(s) will learn how to present their data as publication quality figures, and discuss their research in a clear and concise written format. The student(s) will be exposed to a variety of biochemistry and molecular biology techniques that are applicable to a broad range of experimental contexts. Although the exact techniques performed will depend on the experimental pipeline and research progression, the student(s) can anticipate to learn: 1. Molecular cloning (PCR, mutagenesis, restriction digestion, ligation, seamless cloning, sequence analysis) 2. Bacterial cell culture (pouring plates, transformation, plasmid purification) 3. Protein expression and purification 4. Biophysical analysis (AUC and MALS‐SEC) 5. Human cell culture (routine growth and maintenance, transfection, protein extraction) 6. Immunoprecipitation 7. Western blotting 8. Assessing DNA repair phenotypes of mutant human cell lines MARKING SCHEME (assignments with weight and due date): 1. Research Project Proposal (10%, due 3‐4 weeks after starting the project): Students will provide a 2‐4 page written summary (double‐spaced plus figures and references in standard journal format) of the research project to be conducted. This will include a review of the relevant literature (background), hypothesis, objectives, methods, and significance of the proposed research. 2. Midterm Project Presentation (10%, due mid‐July or mid‐October) Students will give a 20‐minute presentation of their research objectives, results, challenges and future goals to the lab. This will be followed by a 10‐minute discussion period, in which the student will be evaluated on their ability to answer questions related to their research project. 3. Final Research Report (20%, due at noon on the last day of term) Students will provide a 5‐10 page written report (double‐spaced plus figures and references in standard journal format) that describes the background and rationale for the research project, experimental methods, results and interpretation, conclusions and future directions. 4. Attendance, work ethic and participation in lab meetings (15%, ongoing) 5. Lab work (20%, ongoing) Through weekly meetings with Dr. Wyatt, students will be evaluated on their ability to master experimental techniques, as well as their scientific reasoning (data analysis and interpretation, troubleshooting). 6. Lab notes (20%, ongoing) Through guidance and weekly meetings with Dr. Wyatt, students will be evaluated on their ability to maintain high‐quality lab notes that detail all experimental aims, methods, results and interpretation. 7. Lab safety and organization (5%, ongoing) Students will be examined on their knowledge of general lab safety procedures through ‘pop‐up’ questions throughout the term (Dr. Wyatt will provide relevant documents at the start of the term). Students will also be evaluated on their ability to create and maintain a safe, tidy and well‐organized work area. Project Code: BCH 6S RESEARCH OPPORTUNITY PROGRAM 299Y/399Y PROJECT DESCRIPTIONS 2017‐2018 SUMMER Name and Title: Dr. Haley Wyatt, Assistant Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: Using CRISPR/Cas9 to engineer SLX4 proteins in human cells NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: Background Human cells contain sophisticated networks to repair DNA damage and safeguard genome integrity. These pathways ensure proper cell function and help protect us from developing diseases like cancer. Most DNA repair pathways rely on ‘molecular scissors’ called structure‐selective endonucleases (SSEs) to remove toxic DNA structures that form during DNA repair (and normal cellular growth). In human cells, a heterodimeric SSE called SLX1‐SLX4 is critical for DNA repair and genome stability. The biological importance of SLX4 is underscored by the fact that mutations in SLX4 are associated with Fanconi anemia (FA), a rare genetic disease characterized by genome instability and cancer predisposition. Interestingly, the SLX4 subunit interacts with many DNA repair proteins. The prevailing model is that SLX4 provides a hub for the assembly of versatile protein complexes that have distinct functions in the cell. My lab aims to understand the biological and biochemical mechanisms by which SLX4 complexes mediate genome stability. This project will harness the power of CRISPR/Cas9 methodology to engineer human SLX4 proteins that provide us with tools for the complete characterization of endogenous SLX4 complexes. The long‐term goal of this project is to identify novel SLX4‐interacting proteins that provide new insights into the functions and regulation of SLX4 complexes in human cells. Objectives The goals of this research project are two‐fold: 1. Use CRISPR/Cas9 to engineer SLX4 proteins (called STREP‐FLAGSLX4) in model human fibroblasts 2. Optimize affinity purification protocol for STREP‐FLAGSLX4 Methodology With guidance from Dr. Wyatt, the student will use the CRISPR/Cas9 platform to engineer endogenous SLX4 proteins that contain a dual StrepTactin‐Flag affinity tag (STREP‐FLAGSLX4) in model human fibroblasts (HEK 293T). The student will then optimize a protocol for the enrichment of STREP‐FLAGSLX4 complexes from HEK 293T cells. If time permits, the student will perform a large‐scale production of STREP‐FLAGSLX4 complexes for mass spectrometry‐based characterization of the SLX4 ‘interactome’. DESCRIPTION OF STUDENT PARTICIPATION: The student will be directly supervised by Dr. Wyatt, and will be involved in all aspects of the project detailed above. With guidance from Dr. Wyatt, the student will be expected to read appropriate background literature. Dr. Wyatt and a research technician will provide experimental planning and training. The student will be expected to conduct experiments on a semi‐independent basis, with the expectation that they will become more independent as the project progresses. The student will be responsible for keeping accurate and complete records of their experimental lab work. Dr. Wyatt will review these records on a weekly basis. Dr. Wyatt will also provide weekly feedback on the student’s progress. The student will have ample opportunity to learn and/or enhance their communication skills through weekly meetings with Dr. Wyatt, participation in lab meetings, and interactions with neighboring researchers. The student will learn how to present their data as publication quality figures, and discuss their research in a clear and concise written format. The student will be exposed to a variety of biochemistry and molecular biology techniques that are applicable to a broad range of experimental contexts, which include: 1. Molecular cloning (PCR, restriction digestion, ligation, seamless cloning, sequence analysis) 2. Bacterial cell culture (pouring plates, transformation, plasmid purification) 3. Human cell culture (routine growth and maintenance, transfection, protein extraction) 4. Affinity purification 5. Western blotting MARKING SCHEME (assignments with weight and due date): 1. Research Project Proposal (10%, due 3‐4 weeks after starting the project): Students will provide a 2‐4 page written summary (double‐spaced plus figures and references in standard journal format) of the research project to be conducted. This will include a review of the relevant literature (background), hypothesis, objectives, methods, and significance of the proposed research. 2. Midterm Project Presentation (10%, due mid‐July or mid‐October) Students will give a 20‐minute presentation of their research objectives, results, challenges and future goals to the lab. This will be followed by a 10‐minute discussion period, in which the student will be evaluated on their ability to answer questions related to their research project. 3. Final Research Report (20%, due at noon on the last day of term) Students will provide a 5‐10 page written report (double‐spaced plus figures and references in standard journal format) that describes the background and rationale for the research project, experimental methods, results and interpretation, conclusions and future directions. 4. Attendance, work ethic and participation in lab meetings (15%, ongoing) 5. Lab work (20%, ongoing) Through weekly meetings with Dr. Wyatt, students will be evaluated on their ability to master experimental techniques, as well as their scientific reasoning (data analysis and interpretation, troubleshooting). 6. Lab notes (20%, ongoing) Through guidance and weekly meetings with Dr. Wyatt, students will be evaluated on their ability to maintain high‐quality lab notes that detail all experimental aims, methods, results and interpretation. 7. Lab safety and organization (5%, ongoing) Students will be examined on their knowledge of general lab safety procedures through ‘pop‐up’ questions throughout the term (Dr. Wyatt will provide relevant documents at the start of the term). Students will also be evaluated on their ability to create and maintain a safe, tidy and well‐organized work area.