Proteasome Active Sites

In the first experiment, treatment of murine thymus extracts with fluorescent peptide epoxyketone MVB-003 followed by SDS-PAGE revealed, next to the expected bands denoting the catalytic residues of constitutive proteasomes and 

Workflow for the identification of the thymoproteasome p5t active site peptide. 

Remove the excess of probe by chloroform/methanol precipitation. 

Denature the proteins, and open disulfide bond by DTT (dithiothreithol), and then alkylate the cysteines with iodoacetamide. 

Pull down with paramagnetic beads Wash away specific binding proteins On-bead digestion by trypsin Elution of the active site peptides 

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F. Kisselev et al., Proteasome active sites allosterically regulate each other, suggesting a cyclical bite-chew mechanism for protein breakdown. Mol Cell. 4 (1999) 395-402. 

Most of the proteasome ABPs reported are based on either peptide vinyl sulfones or peptide epoxyketones. In Figure 12.3a, the mechanism by which N-terminal threonines within proteasome active sites catalyze peptide bond hydrolysis is depicted. Aligning of either a vinyl sulfone or an epoxyketone at the appropriate position (i.e., the carbonyl of the scissile amide bond) allows covalent and irreversible reaction with the N-terminal threonine-OH (and -NH2 in case of epoxyketones) (Figure 12.3b) and therefore employment of these electrophiles in ABP design. Of the two electrophiles, especially the epoxyketone - evolved in nature - is an intriguing electrophilic trap it presents two electrophilic carbons to the 1,2-aminoalcohol characteristic and almost unique for proteasome active sites. 

Treatment of either a tissue culture or a cell extract with Bodipy-epoxomicrn MVB-003 followed by SDS-PAGE readily reveals proteasome active sites (Figure 12.3c) [9, 10]. In case the constitutive proteasome is expressed exclusively, the three catalytic species pi, P2, and pS are readUy resolved. In case the treated tissue also expresses immunoproteasomes, the one-dimensional gel wUl resolve pi and p2i, but only partially pi, pii, pS, and pSi (see for a detailed experimental protocol Box 12.1 [11]). Two-dimensional gel electrophoresis, by which proteins are separated on the basis of the charge followed by mass allows for complete resolution of all six catalytic activities. Figure 12.3d represents an example of competitive ABPP. In this experiment, tissue or tissue extract is treated first with a prospective inhibitor. Ensuing incubation with... 

Threonine peptidases (and some cysteine and serine peptidases) have only one active site residue, which is the N-terminus of the mature protein. Such a peptidase is known as an N-terminal nucleophile hydrolase or Ntn-hydrolase. The amino group of the N-terminal residue performs the role of the general base. The catalytic subunits of the proteasome are examples of Ntn-hydrolases. 

Proteasomal inhibition represents a novel strategy in cancer treatment and the small molecule Bortezomid (PS-341, Velcade ) has been approved for the treatment of refractory and relapsed multiple myeloma, a proliferative disease of plasma cells. Bortezomid inhibits an active site in a proteasome subunit and remarkably shows selective cytotoxicity to cancer cells. Although the underlying mechanisms are not completely understood bortezomid apparently induces a cell stress response in these tumor cells followed by caspase-dependent apoptosis. Whether bortezomid is beneficial for the treatment of other proliferative disease is currently being tested in clinical trials. 

ASBA has been used to identify parasite adhesive proteins (Gowda et al., 2007), for active site-directed labeling of glucosidase I (Romaniouk et al., 2004), and to study interactions with the proteasome (Qureshi et al., 2003). 

Fig. 4.1. Fundamentals of the ubiquitin system. Adapted from Ref [5]. Figure 4.1 shows the fundamentals of the ubiquitin system. (1) Ubiquitin is synthesized in linear chains or as the N-terminal fusion with small ribosomal subunits that are cleaved by de-ubiquitylating enzymes to form the active protein. Ubiquitin is then activated in an ATP-dependent manner by El where a thiolester linkage is formed. It is then transthiolated to the active-site cysteine of an E2. E2s interact with E3s and with substrates and mediate either the indirect (in the case of HECT E3s) or direct transfer of ubiquitin to substrate. A number of factors can affect this process. We know that interactions with Hsp70 can facilitate ubiquitylation in specific instances and competition for lysines on substrates with the processes of acetylation and sumoylation may be inhibitory in certain instances. (2) For efficient proteasomal targeting to occur chains of ubiquitin linked internally through K48 must be formed. This appears to involve multiple...

Boeodovsky, a., Kessler, B. M., Casageande, R., Oveekleeet, H. S., Wilkinson, K. D., and Ploegh, H. L. A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14, EmboJ, 2001, 20, 5187-96. 

Whereas standard proteases use serine, cysteine, aspartate, or metals to cleave peptide bonds, the proteasome employs an unusual catalytic mechanism. N-terminal threonine residues are generated by self-removal of short peptide extensions from the active yS-subunits and act as nucleophiles during peptide-bond hydrolysis [23]. Given its unusual catalytic mechanism, it is not surprising that there are highly specific inhibitors of the proteasome. The fungal metabolite lactacystin and the bacterial product epoxomicin covalently modify the active-site threonines and in-... 

A new proteasome activator called PA200 was recently purified from bovine testis [156]. Human PA200 is a nuclear protein of 1843 amino acids that activates all three catalytic subunits with some preference for the PGPH active site. Homologs... 

Bogyo, M. et al. Covalent modification of the active site threonine of proteasomal f3 subunits and the Escherichia coli homolg HsIV by a new class of inhibitors. Proc. Natl. Acad. 

Rubin, D. M., Glickman, M. H., Larsen, C. N., Dhruvakumar, S., and Finley, D. Active site mutants in the six regulatory particle ATPases reveal multiple roles for ATP in the proteasome. EMBO J. 1998, 17, 4909-4919. 

Arendt, C. S. and Hoghstrasser, M. Eukaryotic 20S proteasome catalytic subunit propeptides prevent active site inactivation by N-terminal acetylation and promote particle assembly. Embo J 1999, 18, 3575-85. 

All 12 active sites of HslV are located on the inner walls of the hollow particle. In the E. coli particle, each active site has neighboring active sites 28 A away on the same ring and 22 A and 26 A away on the opposite ring. The environment of the nucleophilic Thrl looks similar to that in proteasomes, and the presence of a (putatively protonated) lysine residue near the active site probably helps to lower the pKa of the N-terminal a-amino group so that it is present in the unprotonated form, which can act as the general base to accept a proton from Thrl. 

Fig. 10.5. Molecular surface of the archaeal (A), the euka otic 20S (B) and the HsIV proteasome (C). The accessible surface is colored in blue, the clipped surface (along the cylinder axis) in white. To mark the position of the active sites, the complexes are shown with the bound inhibitor calpain (yellow). (A) The disorder of the first N-terminal residues in the archaeal a-subunits generates a channel in the structure of the CP, (B) whereas the asymmetric but well-defined arrangement of the a N-terminal tails seals the chamber in eukaryotic CPs. (C) The eubacterial "miniproteasome" has an open channel through which unfolded proteins and small peptides can access the proteolytic sites. (D) Ribbon plot of the free...

Fig. 10.7. Inh ibitor binding to individual active sites of the yeast 20S proteasome. The inhibitors lactacystin (A), epoxomicin (B) and TMC95A (C) are colored green and are shown in stereo mode together with their unbiased electron densities. The active-site Thrl is highlighted in black. (A) Covalent binding of the Streptomyces metabolite lactacystin to the active site of 5. The SI pockets of the active subunits and differ from that of 5 and are not suitably constructed to bind the inhibitor. As discussed in the text, Met45 (black), which is located at the bottom of the 5-Sl pocket, makes the difference for inhibitor...

Bogyo, M., McMaster, J. S., Gaczinska, M., Toetoeelia, D., Goldberg, A. L., and Ploegh, H. Covalent modification of the active site threonine of proteasomal p-subunits and the Escherichia coli homolog HslV by a new class of inhibitors. Proc. Nat. Acad. Sci. USA 1997, 94, 6629-6634. 

Bogyo, M., Shin, S., McMastee, J. S., and Ploegh, H. L. Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes. Chem Biol 1998, 5, 307-320. 

Heinemeyer, W., Fischer, M., Krimmer, T., Stachon, U., and Wolf, D. H. The active sites of the eukaryotic 20S proteasome and their involvement in subunit precursor processing. J. Biol. Chem. 1997, 272, 25200— 25209. 

Jager, S., Groll, M., Huber, R., Wolf, D. H., and Heinemeyer, W. Proteasome beta-type subunits unequal roles of propeptides in core particle maturation and a hierarchy of active site function./. Mol. Biol. 1999, 291, 997-1013. 

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