PACMANS: A bioinformatically informed algorithm to predict,design, and disrupt protease‐on‐protease hydrolysis

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.     

Computational predictions of cysteine cathepsin‐mediated fibrinogen proteolysis

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 α, β, and γ 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.     

Autophagy during beef aging

The conversion of muscle into meat is a complex process of major concern for meat scientists due to its influence on the final meat quality. The aim of this study was to investigate the occurrence of autophagic processes in the conversion of muscle into meat. Our findings demonstrated, for the first time, the occurrence of autophagic processes in the muscle tissue at early postmortem period (2 h to 24 h) in both beef breeds studied (Asturiana de los Valles and Asturiana de la Montaña) showing significant time-scale differences between breeds, which could indicate a role of this process in meat maturation. These breeds have different physiological features: while Asturiana de los Valles is a meat-specialized breed showing high growth rate, an elevated proportion of white fibers in the muscle and low intramuscular fat level, Asturiana de la Montaña is a small- to medium-sized rustic breed adapted to less-favored areas, showing more red fibers in the muscle and a high intramuscular fat content.     

Menadione-induced apoptosis in U937 cells involves Bid cleavage and stefin B degradation

Earlier studies showed that the oxidant menadione (MD) induces apoptosis in certain cells and also has anticancer effects. Most of these studies emphasized the role of the mitochondria in this process. However, the engagement of other organelles is less known. Particularly, the role of lysosomes and their proteolytic system, which participates in apoptotic cell death, is still unclear. The aim of this study was to investigate the role of lysosomal cathepsins on molecular signaling in MD-induced apoptosis in U937 cells. MD treatment induced translocation of cysteine cathepsins B, C, and S, and aspartic cathepsin D. Once in the cytosol, some cathepsins cleaved the proapoptotic molecule, Bid, in a process that was completely prevented by E64d, a general inhibitor of cysteine cathepsins, and partially prevented by the pancaspase inhibitor, z-VAD-fmk. Upon loss of the mitochondrial membrane potential, apoptosome activation led to caspase-9 processing, activation of caspase-3-like caspases, and poly (ADP-ribose) polymerase cleavage. Notably, the endogenous protein inhibitor, stefin B, was degraded by cathepsin D and caspases. This process was prevented by z-VAD-fmk, and partially by pepstatin A-penetratin. These findings suggest that the cleaved Bid protein acts as an amplifier of apoptotic signaling through mitochondria, thus enhancing the activity of cysteine cathepsins following stefin B degradation.     

Stimulatory effect of vitamin C on autophagy in glial cells

Intracellular accumulation of damaged or abnormal proteins is a common event associated with numerous neurodegenerative diseases and other age-related pathologies. Increasing the activity of the intracellular proteolytic systems normally responsible for the removal of these abnormal proteins might be beneficial in lessening the severity or development of those pathologies. In this study we have used human astrocyte glial cells to investigate the effect of vitamin C (ascorbate) on the intracellular turnover of proteins. Supplementation of the culture medium with physiological concentrations of vitamin C did not affect protein synthesis, but did increase the rate of protein degradation by lysosomes. Vitamin C accelerated the degradation of intra- and extracellular proteins targeted to the lysosomal lumen by autophagic and heterophagic pathways. At the doses analyzed, vitamin C lowered and stabilized the acidic intralysosomal pH at values that result in maximum activation of the lysosomal hydrolases.     

Autophagy: Many paths to the same end

Different mechanisms lead to the degradation of intracellular proteins in the lysosomal compartment. Activation of one autophagic pathway or another, under specific cellular conditions, plays an important role in the ability of the cell to adapt to environmental changes. Each form of autophagy has its own individual characteristics, but it also shares common steps and components with the others. This interdependence of the autophagic pathways confers to the lysosomal system, both specificity and flexibility on substrate degradation. We describe in this review some of the recent findings on the molecular basis and regulation for each of the different autophagic pathways. We also discuss the cellular consequences of their interdependent function. Malfunctioning of the autophagic systems has dramatic consequences, especially in non-dividing differentiated cells. Using the heart as an example of such cells, we analyze the relevance of autophagy in aging and cell death, as well as in different pathological conditions. (Mol Cell Biochem 263: 55–72, 2004)