Mark T. Fisher, Ph.D.
Department of Biochemistry and Molecular Biology
University of Illinois, Urbana IL. Ph.D., 1987
National Institutes of Health, National Heart, Lung and Blood Institute, Bethesda, Maryland (Staff Fellow)
Publications: Click here
In Memory: Mark T. Fisher
June 21, 1954 - September 4, 2018
It is with great sorrow that we announce the unexpected passing of
our colleague, Dr. Mark T. Fisher.
Dr. Fisher joined the faculty of KU School of Medicine in 1994 and
rose to the rank of Professor in the Department of Biochemistry and Molecular
biology. Dr. Fisher was an active and internationally recognized expert on
protein folding and structure. He was supported through a long history of
extramural grants and partnerships with the NIH and biotech industry.
Mark was a cherished colleague, beloved for his scientific acumen
as much as for his personal warmth and sense of humor. He embodied the role of
Professor: he sought to understand the natural world and his love for this
endeavor was expressed by enthusiastically sharing his knowledge with those
around him, whether trainee or colleague. He was committed to the educational
programs and research enterprise of KUMC and spoke with great conviction in
public and in committees to defend and nurture those missions. In his
professional life, Mark's first commitment was always to his students, and he
reveled in his interactions with graduate and medical trainees. His great love
for science animated him and he safeguarded this treasure by training
generations of scientists and physicians, sharing with them his passion,
knowledge and insights. His professional legacy will persist in his important
body of work, the many trainees whose lives he touched, and the colleagues who
were better scientists and people for knowing him. His departmental colleagues
will truly miss seeing Mark in his riding clothes and helmet, rolling his
bicycle through the department halls - perhaps sunburnt, drenched by rain,
frozen by snow - but always ready with a hearty greeting and a warm smile. He
was a friend to all who knew him and the great sadness born of his absence is a
testament to the life he lived so very well.
The only thing Mark loved more than science was his family.
He was a devoted and loving husband, father and grandfather. He is
survived by his wife Kathleen, three children and grandchildren.
JOVE video here
Video highlight from 2014 publication (scroll down) here.
Alexandra J. Machen, Narahari Akkaladevi, Caleb Trecazzi, Pierce T. O'Neil, Syaranta Mukherjee, Yifei Qi, Rebecca Dillard, Wonpil Im, Edward P. Gogol, Tommi A. White, and Mark T. Fisher (2017) "Asymmetric Cryo-EM Structure of Anthrax Toxin Protective Antigen Pore with Lethal Factor N-Terminal Domains", Toxins 9(10. pii:E298. doi:10.3390/toxins9100298
Pace, SE., Joshi, SB., Esfandiary, R., Bishop, S., Middaugh, CR., Fisher MT.*, Volkin, DB.*, (2018) (*-co corresponding authors). The Use of GroEL-BLI Biosensor to Rapidly assess "Pre-Aggregate" Populations for Antibody Solutions Exhibiting Different Stability Profiles, 2017, J Pharm Sci. 2018
Pierce T. O'Neil,†, Alexandra J. Machen1,†, Benjamin Deatherage, Caleb Tracazzi, Alexander Tischer, Matthew T. Auton, Michael Baldwin, Tommi Ann White, and Mark T. Fisher,* (2018) "The Chaperonin GroEL: A Versatile Tool for Applied Biotechnology Platforms" †cofirst authors, *corresponding author. Frontiers in Molecular Biosciences, 5, article 46, 1-18. doi.org/10.3389/fmolb.2018.00046
Machen, A. J., O'Neil, P. T., Pentelute, B. L., Villar, M.T., Artigues, A., Fisher, M.T. Analyzing Dynamic Protein Complexes Assembled On and Released From Biolayer Interferometry Biosensor Using Mass Spectrometry and Electron Microscopy. J. Vis, Exp. (138), e57902, doi:10.3791/57902 (2018). See above for video link.
Major Research Interests
Projects: Structural biophysics of transient and captured protein states (Anthrax Toxin Pore translocation complex), Protein Folding and Pharmaceutical Drug Development.
Introduction to our research:
Our understanding of protein folding inside the cell has advanced significantly over the last two decades. Biochemists are now aware that cellular protein folding is, in most cases, assisted by other essential proteins called molecular chaperones. With this new knowledge, we are beginning to appreciate the critical role the protein homeostasis plays in cell viability and Human disease. From a medical standpoint, understanding cellular folding is extremely important because valid estimates gleaned from molecular genetic databases indicate that between 30 TO 50% OF THE TOTAL NUMBER OF HUMAN DISEASES AT ONE TIME ARE CAUSED BY SOME SORT OF PROTEIN FOLDING DEFECTS. Among the most well-known folding diseases are Alzheimer's disease, Parkinson's disease, Huntington's, ALS, and Cystic Fibrosis. In addition these more recognizable diseases, we are now also aware that bacterial/viral protein toxins have to undergo folding changes that result in disease. In these cases, numerous bacterial/viral protein toxins (Anthrax, Diphtheria, Botulinum, Ricin, Shiga toxin, influenza, MRSA) literally change folding shapes during infection.
To make any headway on these broad disease fronts, our research efforts must advance from the preliminary discovery phase obtained from genomics or proteomics efforts toward the application to devise targeted therapies to tackle each of the disease states one protein at a time. In order to develop small molecule stabilizers for proteins, we use the high affinity form of the chaperonin protein to detect partially folded proteins (dynamic or metastable). We use this interaction to construct a moderate throughput chaperonin based platform technology using label free technologies Surface Plasmon Resonance (SPR) and Biolayer interferometry (BLI) to screen and validate chemical compound libraries. The latter label free method is the primary platform that we routinely use in the laboratory. The research in the Fisher lab is focused on taking the vast knowledge describing molecular chaperone function and applying this data to establish broad based research tools and approaches to eventually aid in the identification and design of the next generation of small molecule protein drugs to ameliorate Protein Folding Diseases. We have also designed novel protein toxin based platforms to recapitulate and detect endosomal like unfolding/refolding transitions of occur as bacterial or viral toxin gain entry through cellular endosomal membranes.
Major research projects:
The Spear of Anthrax Comes into View. EM structure of the Anthrax Toxin Pore translocon.
The Fisher laboratory along with Ed Gogol from University of Missouri Kansas City and R. John Collier and colleagues from Harvard have constructed the first structural images of the anthrax pore translocon "spear" inserted into a nanodisc lipid bilayer. This pore initially binds cell surface receptors as benign lethal toxin-prepore complexes. The cell then engulfs this trojan horse, transforming the prepore into the pore, allowing the passenger toxins (lethal factor and edema factor) to unfold and thread through the pore interior to disrupt cellular function. The trick to obtain this structure followed Nature's lead which entailed tethering the prepore-lethal toxin complex, followed by transformation to the pore before assembling the nanodisc around the hydrophobic pore tip. The EM data was collected at the NRAMM (National Resource for Automated Molecular Microscopy) Scripps facility (Akkaladevi Protein Science) with Brigit Carragher and at the NCMI (National Center for Macromolecular Imaging) Baylor facility (S. Ludtke and W. Chiu).
Schematic procedure for constructing purified Anthrax translocon pores inserted into lipid nanodiscs.
Akkaladevi et al., (2013) Protein Sci. 22:492-501.
Our structure work with the Protective antigen pore translocon has yielded the first low resolution structures of this membrane protein inserted into lipid nanodiscs using single particle analysis with images from negative stain EM and cryoEM. The figure below shows the progressive improvements for obtaining the structure of the anthrax toxin pore translocon (Protective antigen) using Electron microscopy single particle analysis.
Even these low resolution EM structures reveal some important structural features that lead to some functional insight. For example, the positioning of the phe clamp loop (discovered by Krantz and Collier) appears to be positioned to constrain the opening of the lumen at pH 7.0. In addition, the interior lumen (and exterior) of the anthrax pore translocon does not appear to be a smooth barrel and displays various constrictions, bulges and vestibules. By taking account of its complex electrostatic interior, our next goal use molecular dynamic approaches (MMFF) to model the interior lumen and exterior barrel surface (continuing collaboration with Srayanta Mukherjee, Wonpil Im).
Recapitulating Endosomal Transitions on Label free surfaces :
We have also used the label free surface Plasmon resonance (SPR) and biolayer interferometry (BLI) platforms to measure real time unfolding-refolding transitions of this pore translocon. We constructed the complete anthrax toxin complex using the same methodology we used to construct and purify PA pores inserted into nanodiscs. With the label free surfaces, the SPR or BLI signals can detect the real time transitions of the prepore to pore transitions (see figure below for BLI). When the partial anthrax toxin is constructed on the label free surfaces (e.g. attached LFN-PA prepore), the acidification of the solution mimicks the late endosomal pH (5.0) and results in the transition of the prepore to pore conformation.
The figure to the right illustrates the observed signal response that arises from the PA prepore to pore transition alone. The use of biolayer interferometry biosensor tips allowed us to remove the transitioned Pore translocons from the surface and transfer these to Electron microscopy grids (bottom of the figure) to view the transitioned pore. The presence of a lipid micelle shows that the pores no longer aggregate and can easily be resolved as soluble individual pores.
Designing Chaperonin-Based HTS platforms to identify Protein folding disease stabilizers
Identifying pharmacological chaperones: A number of protein folding diseases develop because missense mutations shift the equilibrium distribution between native and partially denatured species. In addition to missense mutation induced changes, equilibrium shifts toward misfolded protein populations also can result from changes in the solution environment or through the simple overexpression of the native protein. An emerging strategy and potential therapy for ameliorating some of these misfolding diseases is to employ so called "pharmacological chaperones" or more appropriately, protein stabilizers. These stabilizers or chemical chaperones bind and stabilize native states in equilibrium with disease causing states and prevent, through mass action effects, the accumulation of the deleterious misfolded protein. We are now developing label-free systems to rapidly identify and test for plausible pharmacological chaperones. As illustrated below, the chaperonin will kinetically bind and partition partially folded states of proteins. If a stabilizer is present, this partitioning is decreased or is no longer observed. This is the basis of our generic screening procedure.
"Using Natures sentries to capture the seeds of destruction:".
Naik, et al., "On the Design of Broad Based Screening Assays to Identify Potential Pharmacological Chaperones of Protein Misfolding Diseases." Current Opinions in Medicinal Chemistry vol 12 (22): 2504-22.
User Presentations for fortébio (a division of Pall Life Sciences)
M Fisher presentation at ~ 20 min into Webinar.