Aspartate-directed proteases

The stress or growth pathways modulated by vanadium involve specialized effectors and often can be activated by excess ROS. Cytokines, small proteins that effect communication between cells or cell behavior, can be involved in the cellular stress response. Tumor necrosis factor a (TNFa) is a cytokine stress signal that binds to a membrane receptor (tumor necrosis factor receptor, or TNFR). This interaction stimulates kinase activity that leads to cell injury and inflammation and also to the activation of caspases, a family of cysteine-dependent aspartate-directed proteases that are involved in apoptosis. The mitogen-activated protein (MAP) kinase cascade regulates both mitosis and apoptosis signaling pathways. 

Aspartate-directed proteases are a family of at least seven similar enzymes, including Granzyme B (also known as Fragmentin 2 or CPPl), which... 

Figure 1.18 Left Piperidine-derived inhibitors (top) were shown to inhibit aspartic acid proteases by bridging the two catalytically active aspartate residues (bottom). Based on this finding [99, 100], together with the knowledge of the cyclohexanone-based active site-spanning cysteine protease inhibitors [96-98], novel cyclic warheads against aspartic acid proteases can be designed as target family-directed privileged structures [3, 101],...

Kick, E.K. and Ellman, J.A., Expedient method for the solid-phase synthesis of aspartic acid protease inhibitors directed toward the generation of libraries, J. Med. Chem., 38 (1995) 1427-1430. 

While catalysis by aspartic proteases involves the direct hydrolytic attack of water on a peptide bond, catalysis... 

Figure 7-6. Mechanism for catalysis by an aspartic protease such as HIV protease. Curved arrows Indicate directions of electron movement. Aspartate X acts as a base to activate a water molecule by abstracting a proton. The activated water molecule attacks the peptide bond, forming a transient tetrahedral Intermediate. Aspartate Y acts as an acid to facilitate breakdown of the tetrahedral intermediate and release of the split products by donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its initial state.

A. A. Kossiakoff, S. A. Spencer, Direct Determination of the Protonation States of Aspartic Acid-102 and Histidine-57 in the Tetrahedral Intermediate of the Serine Proteases Neutron Structure of Trypsin , Biochemistry 1981, 20, 6462-6474. 

The presence of tumor necrosis factor a directly leads to apoptosis via interaction with the tumor necrosis factor receptor, one of a class of receptors referred to as death receptors. NF-kB, which must enter the nucleus to initiate apoptosis, is a transcription factor sequestered in the cytoplasm by inhibitor of kB (IkB). The binding of TNFa to its receptor leads to the ubiquitin-dependent proteolysis of IkB, allowing NF-kB to enter the nucleus. The activation of apoptosis results directly from the stimulation of NF-kB, a transcription factor whose phosphorylation is controlled by vanadium compounds. In a global gene expression study, it was found that diabetes increased the formation of IkB, whereas vanadium compound treatment lowered the production of this inhibitor [101]. The activation of the TNFR also activates the caspase proteins, a class of proteases that cleave proteins after specific aspartate residues. 

Effector caspases are activated by a transactivation mechanism, which is characterized by the catalytic action of a mature caspase on a procaspase (Thornberry et al., 1997 Earnshaw et al., 1999 Slee et al., 1999). Nevertheless, their activation can also occur by the action of other proteases. Granzyme B, a serine-protease, also has proteolytic specificity for aspartic acid residues. It is able to cleave and directly activate caspase 3 (Darmon et al., 1995). Cathepsin B, a lysosomal protease, cleaves and activates procaspase 11 (Schotte et al., 1998). 

FIGURE 32. Crystal structure of dimeric HIV protease with the bound inhibitor tipranavir (Q7K) viewed from the direction perpendicular to the pseudo-twofold axis, wherein the enoUc 4-hydroxyl functionality is H-bonded to the active-site aspartate side chains (Plate LX)... 

Figure 8 Comparison of signal peptide peptidase (SPP) with presenilin and the y-secretase complex. Signal peptides are removed from membrane proteins via signal peptidase (SP), and these peptides are released from the membrane by SPP-mediated intramembrane proteolysis. SPP, like presenilin, contains two aspartates that are essential for protease activity, but the conserved aspartate-containing motifs are in the opposite orientation compared with their presenilin counterparts. Consistent with the flipped orientation of SPP vis-a-vis presenilin, the substrates of these two proteases also run in the opposite direction. Unlike presenilin, SPP apparently does not require other protein cofactors or cleavage into two subunits for proteolytic activity.

Recent achievements in the development of active-site directed affinity probes for proteases and other enzyme classes provide direct chemical labeling of proteases of interest in the biological system (24-27). These specific activity probes allow joint evaluation of selective protease inhibition concomitant with labeling of relevant protease enzymes for more analyses. Moreover, activity-based probes that selectively label the main protease subclasses—cysteine, serine, metallo, aspartic, and threonine—can provide advantageous chemical approaches for functional protease identification. Activity probe labeling of proteases allows direct identihcation of enzyme proteins by tandem mass spectrometry. Such chemical probes directed to cysteine proteases have been instrumental for identification of the new cathepsin L cysteine protease pathway for neuropeptide biosynthesis, as summarized in this article. 

The protease exists as a homodimer. Each 99-residue monomer contains 10 j3-strands and the dimer is stabilized by a four-stranded antiparallel jS-sheet formed by the N- and C-terminal strands of each monomer. The active site of the enzyme is formed at the interface, where each monomer contributes a catalytic triad (Asp2 -Thr2 -Gly ) that is responsible for cleavage of the protease substrates. The "flap region" is located above the reactive site and is formed by a hairpin from each monomer of two antiparallel j3-strands joined by a j8-turn. There is little difference between the solution and crystal structures of protease-inhibitor complexes, except in those regions where the polypeptide chain is disordered. However, experiments in solution have allowed access to parameters that are not directly accessible from crystal data. These parameters, such as the amplitude and frequency of backbone dynamics, the protonation states of the catalytic aspartate residues, and the rate of monomer interchange, are essential in understanding the interaction of HIV protease with potent inhibitors. 

A.A. Kossiakoff and S.A. Spencer. 1981. Direct determination of the protonation states of aspartic acid-102 and histidine-57 in the tetrahedral intermediate of the serine proteases Neutron structure of trypsin Biochemistry 20 6462-6474. (PubMed)... 

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