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

University of Kansas Medical Center
913-588-6940
mfisher1@kumc.edu

Latest publications: Three-dimensional structure of the anthrax toxin pore inserted into lipid nanodiscs and lipid vesicles.Katayama H, Wang J, Tama F, Chollet L, Gogol EP, Collier RJ, Fisher MT.Proc Natl Acad Sci U S A. 2010 Feb 8. [Epub ahead of print]

Identifying protein stabilizing ligands using GroEL.Naik S, Haque I, Degner N, Kornilayev B, Bomhoff G, Hodges J, Khorassani AA, Katayama H, Morris J, Kelly J, Seed J, Fisher MT. Biopolymers. 2010 Mar;93(3):237-51.

Major Research Interests

Importance of 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 HUMAN DISEASES ARE CAUSED BY 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 other diseases caused by protein misfolding result in Emphysema, Liver damage, various Cancers, Diabetes, Polycystic Kidney Diseases, cardiomyopathies, and neuropathies as well as an incredibly wide range of metabolic disorders. To make any headway on this broad disease front, our research efforts must advance from the preliminary discovery phase obtained from genomics or proteomics efforts toward the application and mechanistic phases where we can specifically devise therapies to tackle each of the disease states one protein at a time. 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.

Approach:

Nature uses molecular chaperones to prevent or regulate these inherent protein misfolding disease reactions from occurring or at least from occurring early in life. Of all the molecule chaperones that have been studied, we have found that the best chaperone protein that is the most versatile is the Hsp-60 (Heat shock protein-60) or GroEL chaperonin class isolated from Escherichia coli. This particular chaperonin is an evolutionary counterpart to the Hsp-60 chaperonin that is found within all eukaryotic Mitochondria. Currently there are three ongoing research efforts in the Fisher laboratory that exploit the properties of this adaptable chaperone:

Projects: Structural biophysics of transient and captured protein states (Anthrax Toxin Pore translocation complex), Protein Folding and Pharmaceutical Drug Development.

Structure of the folding intermediates or transition protein states bound to GroEL. To elucidate the mechanism and important structural components of chaperonin facilitated folding, we examine the allosteric structural consequences of protein substrates binding to GroEL. We have found that we can capture and visualize and obtain three dimensional structures by electron microscopy, GroEL molecules with bound protein substrates. These bound substrates can either be small partially folded protein substrates or even extremely large proteins >>100 kDa. Our ability to solve intermediate resolution structures (6-13 Å) also allows us to determine the molecular origins of changes in GroEL structure that result in the transmission of protein allosteric signals over 140 Å within the GroEL oligomer.

Even though folding proteins is the most important function of the GroEL system, we are now using the chaperonin GroEL as a structure platform to anchor and determine functionally important, previously hard to resolve, large scale conformational changes in select protein substrates.  Our most successful project to date involves the capture and visualization of the Anthrax toxin pore translocation complex (Protein Antigen Pore) that is involved in tranlocating the other anthrax toxin components, lethal factor and edema factor from the endosome into the cytoplasm.  The pore acts as the needle to inject partially folded toxins into the cell, resulting in cell death.  Our laboratory collaborates with Professor John Collier and Colleagues at the Harvard Medical School.

Capture, stabilization and visualization of the anthrax toxin pore using GroEL as a molecular scaffold. Katayama et al., 2008, Nat. Struc. Mol. Biol.

Insertion of Anthrax Toxin Pore into lipid nanodiscs and resolving structure using negative stain Electron microscopy. Katayama et al., 2010 Proc. Natl Acad. Sci.

 


In another fruitful collaboration, Florence Tama (University of Arizona) and our laboratory have used Normal Mode Flexible Fitting of the X-ray crystallographic coordinates of the PA prepore to localize the position of the phe clamp, an important structure within the lumen of the pore that facilitates pH driven protein translocation. We are expanding our use of electron microscopy  and nanodisc technology to visualize other important bacterial toxins inserted into model membrane bilayers.   Obtaining a anthrax toxin pore translocation has been unsuccessful since 1997.  Our lab is the first in the world to visualize these structures both without and most importantly inserted into nanodiscs.

Refolding structural proteins for drug targeting: In addition to using molecular chaperones as folding aids, nature also enlists the help of various small molecules called osmolytes to protect proteins from denaturation. In 2000, we discovered that combining molecular chaperonins and osmolytes results in a particularly efficient means to fold a whole host of proteins, including proteins that normally misfold in human diseases. We hold a patent on this particular system (called the Chaperonin/osmolyte folding System or COSMOS). We are developing both small and large scale procedures to refold proteins isolated from inclusion bodies (aggregates of largely pure overexpressed target proteins). This process is now highly relevant since large pharmaceutical companies are revisiting approaches that aim to refold proteins from inclusion bodies because refolding proteins with this procedure appears to be more cost effective method to obtain large quantities of pharmaceutically relevant proteins. These particular refolded proteins are often structural targets for drug action, are themselves protein drugs, or are being used to develop protein diagnostics. Our method is particularly attractive because we can rapidly refold proteins at high concentrations and we can reuse our GroEL folding platform using an immobilized system. Furthermore, our COSMO System does not require the full complement of chaperonin proteins (i.e. the smaller molecule co-chaperonin GroES). The basis for the success of this method lies in the fact that the presence of osmolytes forces peptide backbones toward a buried (often times properly folded) state in the interior of the protein. This osmolyte-induced collapse in turn decreases the binding affinity of the GroEL,, thus allowing for a simple release of the folding protein from the chaperonin using ATP.

Scheme of the chaperonin/osmolyte Protein folding array  

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 an easy to use system 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 not observed.  This is the basis of our generic screening procedure.