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Computational predictions of cysteine cathepsin-mediated fibrinogen proteolysis.
Finding Your Inner Modeler IV

Abstract (Expand)

Fibrin clot formation is a proteolytic cascade of events with thrombin and plasmin identified as the main proteases cleaving fibrinogen precursor, and the fibrin polymer, respectively. Other proteases … may be involved directly in fibrin(ogen) cleavage, clot formation, and resolution, or in the degradation of fibrin-based scaffolds emerging as useful tools for tissue engineered constructs. Here, cysteine cathepsins are investigated for their putative ability to hydrolyze fibrinogen, since they are potent proteases, first identified in lysosomal protein degradation and known to participate in extracellular proteolysis. To further explore this, we used two independent computational technqiues, molecular docking and bioinformatics sequence analysis (PACMANS), to predict potential binding interactions and sites of hydrolysis between cathepsins K, L, and S and fibrinogen. By comparing the results from these two objective, computational methods, it was determined that cathepsins K, L, and S do bind and cleave fibrinogen alpha, beta, and gamma chains at similar and unique sites. These differences were visualized experimentally by the unique cleaved fibrinogen banding patterns after incubation with each of the cathepsins, separately. In conclusion, human cysteine cathepsins K, L, and S are a new class of proteases that should be considered during fibrin(ogen) degradation studies both for disease processes where coagulation is a concern, and also in the implementation and design of bioengineered systems.

Authors: M. C. Ferrall-Fairbanks, D. M. West, S. A. Douglas, R. D. Averett, M. O. Platt

Date Published: 22nd Dec 2017

Publication Type: Journal

Investigating the Life Expectancy and Proteolytic Degradation of Engineered Skeletal Muscle Biological Machines.
Finding Your Inner Modeler IV

Abstract (Expand)

A combination of techniques from 3D printing, tissue engineering and biomaterials has yielded a new class of engineered biological robots that could be reliably controlled via applied signals. These … machines are powered by a muscle strip composed of differentiated skeletal myofibers in a matrix of natural proteins, including fibrin, that provide physical support and cues to the cells as an engineered basement membrane. However, maintaining consistent results becomes challenging when sustaining a living system in vitro. Skeletal muscle must be preserved in a differentiated state and the system is subject to degradation by proteolytic enzymes that can break down its mechanical integrity. Here we examine the life expectancy, breakdown, and device failure of engineered skeletal muscle bio-bots as a result of degradation by three classes of proteases: plasmin, cathepsin L, and matrix metalloproteinases (MMP-2 and MMP-9). We also demonstrate the use of gelatin zymography to determine the effects of differentiation and inhibitor concentration on protease expression. With this knowledge, we are poised to design the next generation of complex biological machines with controllable function, specific life expectancy and greater consistency. These results could also prove useful for the study of disease-specific models, treatments of myopathies, and other tissue engineering applications.

Authors: C. Cvetkovic, M. C. Ferrall-Fairbanks, E. Ko, L. Grant, H. Kong, M. O. Platt, R. Bashir

Date Published: 19th Jun 2017

Publication Type: Journal

PACMANS: A bioinformatically informed algorithm to predict, design, and disrupt protease-on-protease hydrolysis.
Finding Your Inner Modeler IV

Abstract (Expand)

Multiple proteases in a system hydrolyze target substrates, but recent evidence indicates that some proteases will degrade other proteases as well. Cathepsin S hydrolysis of cathepsin K is one such … example. These interactions may be uni- or bi-directional and change the expected kinetics. To explore potential protease-on-protease interactions in silico, a program was developed for users to input two proteases: (1) the protease-ase that hydrolyzes (2) the substrate, protease. This program identifies putative sites on the substrate protease highly susceptible to cleavage by the protease-ase, using a sliding-window approach that scores amino acid sequences by their preference in the protease-ase active site, culled from MEROPS database. We call this PACMANS, Protease-Ase Cleavage from MEROPS ANalyzed Specificities, and test and validate this algorithm with cathepsins S and K. PACMANS cumulative likelihood scoring identified L253 and V171 as sites on cathepsin K subject to cathepsin S hydrolysis. Mutations made at these locations were tested to block hydrolysis and validate PACMANS predictions. L253A and L253V cathepsin K mutants significantly reduced cathepsin S hydrolysis, validating PACMANS unbiased identification of these sites. Interfamilial protease interactions between cathepsin S and MMP-2 or MMP-9 were tested after predictions by PACMANS, confirming its utility for these systems as well. PACMANS is unique compared to other putative site cleavage programs by allowing users to define the proteases of interest and target, and can also be employed for non-protease substrate proteins, as well as short peptide sequences.

Authors: M. C. Ferrall-Fairbanks, Z. T. Barry, M. Affer, M. A. Shuler, E. W. Moomaw, M. O. Platt

Date Published: 13th Jan 2017

Publication Type: Journal

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