John Anagli, Ph.D.
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RESEARCH INTERESTS:
Research in the Anagli lab is focused on understanding the pathophysiological role of proteases of medical relevance, and developing novel protease inhibitors for use as research tools and/or therapeutic agents. Of particular interest are the cysteine proteases calpain, cathepsin B and cathepsin L, which have been implicated in postischemic neuronal injury and tumor cell invasion and metastasis.
The calpain system
The calpain-calpastatin proteolytic system is thought to play an important role in cell proliferation, signal transduction and apoptosis. According to recent research, uncontrolled activation of calpain in the central nervous system (CNS) results in neurodegenerative disorders, including tissue damage following stroke, traumatic brain and spine injury. It has also been linked to pathogenesis of a variety of neurological diseases, such as multiple sclerosis, Alzheimer’s, Huntington’s and Parkinson’s disease. The role of calpain in these processes and how the proteinase is regulated in vivo is not clear, underscoring the importance of understanding the mechanism(s) involved. One key to understanding calpain regulation and function is to determine how the endogenous proteinaceous inhibitor calpastatin interacts with calpain to regulate proteolysis.
We have earlier identified two “hot spots” within the calpastatin molecule in which side chains of the most critical residues interact with hydrophobic pockets in calpain. Recent findings from our laboratory suggest that an initial transient low affinity interaction between calpain and the hydrophobic side chains of several residues in calpastatin constitutes a key step in the reaction pathway that involves calpain sensitization to Ca2+ and the concerted conformational changes within the protease and inhibitor that lead to the specific and high affinity binding of the two molecules.
The knowledge derived from the structure-function relations (SAR) studies mentioned above have been translated into the development of a blood-brain-barrier (BBB)-permeant specific inhibitor of calpains. The novel calpastatin peptide-derived inhibitor is able to cross the BBB, penetrate neurons, reduce infarct volume and improve neurological functional recovery after stroke in rats.
Cathepsins B and L
Our previous research on pharmacological inhibition of cathepsins B and L in vivo with peptidyl diazomethyl ketones has resulted in a U.S. Patent No. 6,458,760 for a “Method for Treating Tissue Damaged from Ischemia”. Additional efficacy studies are being pursued to further the research on these compounds and to attract collaborations in the industry.
Biomedical Proteomics
Our laboratory runs the proteomics core facility of Henry Ford Hospital. The core facility provides support for the experimental design of proteomics-related studies, including pilot and feasibility projects. The services provided include high resolution, 2-(multi)dimensional, all-liquid separation of difficult and complex protein mixtures in a customer-supplied sample (ex. tissues, cells, blood, serum, plasma, urine) and fast imaging of protein patterns, profiles or maps – “protein fingerprinting”. A computer software allows for the side-by-side imaging of two related protein maps for differential comparison and expression level change monitoring of, for example, tumor and normal tissue samples. A differential display and analysis with respect to properties such as expression levels, posttranslational modifications, and interactions with other molecules can be performed for specific biological systems and pathways.
Current Lab Personnel
Kadija Abounit (Graduate Student)
Ashkhen Movisiyan (Research Assistant)
Carmen Tugulan (Undergraduate Student)
Some Lab Alumni
Lisa Blum (Co-op and Summer Research Intern 1998-1999), now Fellow (Neo-Perinatology) Motts Children’s Hospital, Ann Arbor, MI
Pamela Osenkowski (Co-op and Summer Intern 1999), now Postdoc (Neurologic Diseases) Harvard Medical School/Brigham and Women's Hospital, Boston, MA
Randa Jaafar (Co-op and Summer Research Intern 2000-2003), now Resident (Anesthesiology), Case Western/University Hospitals, Cleveland, OH
Cyrena Gawuga (Summer Research Intern 2001, 2002), now MD/PhD student, Brown University Medical School, RI
David Ringger (Summer Research Intern 2001), now Graduate Student (Medicinal Chemistry), University of Redding, United Kingdom
Brandon Kakos (Co-op and Summer Intern 2004-05), now Medical Student, Wayne State University School of Medicine, Detroit, MI
Shantel Weinsheimer (Research Assistant 1999–2002), now Postdoc, Children’s Hospital Oakland Research Institute, Oakland, CA
Russell Betts (Postdoc 2000–03), now Instructor (Drug Discovery), Henry Ford Hospital, Detroit, MI
Laura Stewart (Research Assistant 2004–06), Graduate Student, Beckins Dickinson; Johns Hopkins University, Baltimore, MD
Selected Recent Publications
Betts, R., Weinsheimer, S., Blouse, G. and Anagli, J. (2003) Structural Determinants of the Calpain Inhibitory Activity of Calpastatin Peptide B27-WT. J. Biol Chem. 278, 7800-7809
Betts, R. and Anagli, J. (2004) The β- and γ-CH2 of B27-WT’s Leu11 and Ile18 Side Chains Play a Direct Role in Calpain Inhibition. Biochemistry 43, 2596-2604
Komissarov, A.A., Andreasen, P.A., Bodker, J.S., Declerck, P.J., Anagli, J.Y. and Shore, J.D. (2005) Additivity in effects of vitronectin and monoclonal antibodies against alpha-helix F of plasminogen activator inhibitor-1 on its reactions with target proteinases. J Biol Chem. 280(2), 1482-9.
Yin, S., Lockett, J., Meng, Y., Biliran, H., Blouse, G.E., Li, X., Reddy, N., Zhao, Z., Lin, X., Anagli, J., Cher, M.L. and Sheng, S. (2006) Maspin Retards Cell Detachment via a Novel Interaction with the uPA/uPAR System Can. Res. 66, 4173-81
Anagli, J., Abounit, K., Stemmer, P., Han, Y., Allred, L., Weinsheimer, S., Movsisyan, A. and Seyfried, D. (2008) Effects of cathepsin B and L inhibition on postischemic protein alterations in the brain. Biochem. Biophys. Res. Comm. 366, 86-91
