Project Code: BCH 1 RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 FALL/WINTER Name and Title: Dr. Cordula Enenkel (Associate Professor) Department: Biochemistry TITLE OF RESEARCH PROJECT: Studies on Proteasome Storage Granuli NUMBER OF STUDENT PLACES AVAILABLE: 1‐2 OBJECTIVES AND METHODOLOGY: In quiescent cells, many metabolic enzymes including proteasomes self‐assemble into membrane‐less organelles of unknown function. We aim to understand the formation and clearance of these organelles, their catalytic activity and the physiological consequence of their absence. Our high‐throughput genetic array and mass spectrometry analysis in yeast revealed proteins that are candidate scaffolds for proteasome organelles. We will purify these proteins and proteasomes to reconstitute proteasome organelles and to analyze proteasome dynamics. Their structure will be solved by Xray crystallography. DESCRIPTION OF STUDENT PARTICIPATION: The student will purify proteins and proteasomes by affinity chromatography using the ÄKTA system. Gel electrophoresis under native and denaturing conditions will be applied to analyze proteasome assemblies. Direct fluorescence microscopy of GFP‐labelled proteasomes will be used to monitor proteasome dynamics dependent on the availability of scaffold and transporter proteins. The student will gather broad experience in methods used in protein biochemistry, molecular biology and yeast genetics. MARKING SCHEME (assignments with weight and due date): 2 Presentations (30%) due beginning of December 2014 and of April 2015 End term report (30%) due beginning of April 2015 Lab performance (30%) due beginning of April 2015 Research Proposal (10%) due beginning of Nov 2014 Project Code: BCH 2 RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 FALL/WINTER Name and Title: Greg Fairn, Assistant Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: Characterization of Mammalian Aminophospholipid Flippases NUMBER OF STUDENT PLACES AVAILABLE: 1 OBJECTIVES AND METHODOLOGY: A hallmark of eukaryotic cells is the limited exposure of phosphatidylserine and to a lesser extent phosphatidylethanolamine on the outer leaflet of the plasma membrane. This fundamental feature of eukaryotic cells is maintained by the action of aminophosphospholipid translocases aka flippases. The yeast S. cerevisiae has five flippases while the human genome encodes 14 putative flippases. A by‐product of flipping is the generation of membrane curvature that can support the generation of transport vesicles. Specific mammalian flippases are associated with a variety of pathophysiology including intrahepatic cholestasis, insulin resistance, obesity, neuronal degeneration and Alzheimer’s disease. The objective of this project will be to assess the ability of mammalian Golgi localized flippases to functionally replace their yeast homologue(s) and to purify the flippases for biochemical analysis. The student will be trained in a variety of biochemical, molecular and microbiological techniques such as PCR, subcloning, SDS‐PAGE, Western blotting, protein purification and culturing of E. coli and S. cerevisiae. DESCRIPTION OF STUDENT PARTICIPATION: The student will be involved in the cloning of a number of mammalian flippases into expression vectors using PCR and standard molecular biology techniques. The flippases will be heterologously expressed in yeast cells to determine if they can functionally replace previously characterized yeast homologue(s). Growth and vesicular transport assays will be used to verify functionality. If time permits, the human flippases will also be purified from yeast cells using affinity chromatography to determine phospholipid substrates in vitro. The student will work closely with one or two post‐doctoral fellow(s) in my lab located at St. Michael’s Hospital. MARKING SCHEME (assignments with weight and due date): 1. Project Proposal (2 pages plus figures and references, due within ~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 3 RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 FALL/WINTER 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: 1 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 4 RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 FALL/WINTER Name and Title: Dr Warren Lee, Assistant Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: Molecular Determinants of LDL Transcytosis Across the Coronary Endothelium NUMBER OF STUDENT PLACES AVAILABLE: 2 OBJECTIVES AND METHODOLOGY: The accumulation of LDL under the coronary arterial endothelium is the first step in the pathogenesis of atherosclerosis, one of the commonest causes of death in Canada. How LDL exits the vascular lumen and crosses the endothelial monolayer is very poorly understood, in large part due to technical limitations in studying endothelial permeability. Our lab has devised novel assays to measure how LDL crosses the endothelium and is currently delineating the molecular mechanisms. Ultimately this work may lead to novel approaches to both the prevention and treatment of atherosclerosis. The methodology used includes high‐
resolution live cell imaging as well as traditional biochemical and molecular biology techniques. For further details, please consult the lab website (warrenleelab.com) DESCRIPTION OF STUDENT PARTICIPATION: Experience with cell culture is preferred, and enthusiasm and industriousness are essential. Under appropriate supervision, the student will perform primary cell culture, live cell imaging, immunoblotting and immunofluorescence and will be taught analytical and presentation skills. MARKING SCHEME (assignments with weight and due date): 2‐page Interim report 25%, due November 15th (for Fall term) 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 5 RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 FALL/WINTER Name and Title: Trevor Moraes Assistant Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: Structural and Functional Examination of Membrane Protein Interaction Surfaces NUMBER OF STUDENT PLACES AVAILABLE: 1 OBJECTIVES AND METHODOLOGY: The goal of this research is to analyze the interactions between protein components of ion translocation machineries. Using purified proteins, we will examine their interactions using structures obtained by X‐ray crystallography and biophysical methods including; biolayer interferometry (BLI), isothermal titration calorimetry (ITC), surface plasmon resonance (SPR), and affinity capture assays. DESCRIPTION OF STUDENT PARTICIPATION: ROP299 students will be involved in creating site directed mutants of proteins involved in Ion translocation machineries 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, SPR or ITC). Structures of interesting mutant proteins will be determine using crystallography. 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 6
RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 FALL/WINTER Name and Title: Reinhart Reithmeier Professor Department: Biochemistry TITLE OF RESEARCH PROJECT: Structural Biology of Bacterial Anion Transporters NUMBER OF STUDENT PLACES AVAILABLE: 3 OBJECTIVES AND METHODOLOGY: To determine the crystal structure of a bacterial member of the SLC26 (SulP) family of anion transporters. DESCRIPTION OF STUDENT PARTICIPATION: Students will form a team of researchers working under the supervision of the Professor, a technician and a post‐doctoral fellow to clone, express, purify and crystallize bacterial member of the SLC26 family of anion transporters of their own choosing. Must be enrolled in or have complete and introductory course in Biochemistry (BCH242, BCH210 and BCH320). MARKING SCHEME (assignments with weight and due date): Project proposal (Due October 15th) 10% Paper review and presentation (Due before December 15th) 15% Project report and presentation (Due February 15th) 25% Final project report and presentation (Due April 15th) 50% Project Code: BCH 7 RESEARCH OPPORTUNITY PROGRAM 299Y PROJECT DESCRIPTIONS 2014‐2015 FALL/WINTER 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 first week in January 10% 20‐minute presentation in the first week of January 10% work in the laboratory in the first week of January 25% 15‐page report at end of term in April 15% 30‐min presentation at end of term in April 35% work in the laboratory at end of term in April