Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

Wang, Ching C.; Bozdech, Zbynek; Liu, Chao-lin; Shipway, Aaron; Backes, Bradley J.; Harris, Jennifer L.; Bogyo, Matthew

doi:10.1074/jbc.m300195200

Cited by 44 publications

(35 citation statements)

References 41 publications

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“…1c): Mtb20SWT, Mtb20SOG, Rhod20S, and bov20S. Substrate concentrations were kept at 10 M, equal to or a few-fold lower than the values of K m (10 -200 M) previously reported for tri/tetrapeptide substrates (24,30). Under these conditions, reaction rates likely reflected the specific activity (k cat /K m ) in the absence of substrate saturation effects.…”

Section: Ac-p3p2p1-amc Library and Assaymentioning

confidence: 99%

“…Studies of substrate specificities have been reported for proteasomes of human (22,23) and another eukaryote, Trypanosoma brucei (24), using positional scanning substrate or inhibitor libraries. The positional scanning libraries were usually constructed as 20 pools/position (P1, P2, P3, and P4), each of which contained large numbers of peptides fixed at a single residue and otherwise variant.…”

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confidence: 99%

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Distinct Specificities of Mycobacterium tuberculosis and Mammalian Proteasomes for N-Acetyl Tripeptide Substrates

Lin

Tsu

Dick

et al. 2008

Journal of Biological Chemistry

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Mycobacterium tuberculosis (Mtb),4 the single leading cause of death from bacterial infection, is growing even more dangerous with the spread of resistance to the existing chemotherapeutic agents (1). After four decades without the introduction of new anti-tuberculosis agents into the clinic, efforts are underway to find new chemophores that inhibit new targets and shorten the 6 -9-month treatment regimen that makes compliance problematic (2-4). Many authorities believe that this will require inclusion of agents active against nonreplicating Mtb (5-8).Proteasomes provide the major pathway of intracellular protein degradation in eukaryotic cells, where they are essential for adaptation to changing circumstances and avoidance of toxicity from oxidized or denatured proteins (9 -11). Mtb is one of the few bacteria known to express a proteasome (12). The Mtb proteasome is emerging as a potential anti-tuberculosis drug target. It defends the bacterium against host nitrosative stress (12) and is essential for Mtb to persist in mice (13). The Mtb proteasome can be inhibited by small molecules both as a recombinant protein complex (14, 15) and within intact mycobacteria (12). The structure of the Mtb proteasome has been solved with a peptidyl boronate (15). However, for proteasome inhibition to be a viable anti-infective approach, the inhibitors must be much more active against the proteasome of the bacterium than against that of the host. Partial inhibition of host proteasomes has been successful in the treatment of certain malignancies (16 -18). Toxicity in this setting is often considered acceptable but would be highly undesirable in the treatment of an infectious disease. The extensive conservation of proteasome structure makes it challenging to find inhibitors with species selectivity.A standard approach to developing selective protease inhibitors is to attach a warhead to a selective substrate (19). Among proteasome inhibitors, peptidyl boronates exemplify this approach and have achieved clinical utility (16 -18). Thus, we have embarked on a program to develop peptidyl boronates with greater potency for the Mtb proteasome than for the ␤5 subunit of the human proteasome, whose inhibition is chiefly responsible for the toxicity of these compounds (16, 17). As a step toward that goal, the present study sought potentially exploitable differences in the preferences of bacterial and mammalian proteasomes for tripeptide substrates.The proteasome core, here called "20S," is a cylindrical 28-meric protein complex with proteolytic sites shielded within a central chamber (11)

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Section: Ac-p3p2p1-amc Library and Assaymentioning

confidence: 99%

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confidence: 99%

Distinct Specificities of Mycobacterium tuberculosis and Mammalian Proteasomes for N-Acetyl Tripeptide Substrates

Lin

Tsu

Dick

et al. 2008

Journal of Biological Chemistry

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“…High-resolution crystal structures have demonstrated that the PA28 homolog, PA26, induces a conformational change in the N-termini of 20S ␣-subunits that opens the entrance of the catalytic chamber (Whitby et al, 2000;Forster et al, 2005). PA28 (PA26) also alters the proteolytic properties of 20S and/or 19S-20S proteasomes by modifying the pattern, but not the overall size of cleaved products (Harris et al, 2001;Li et al, 2001;Cascio et al, 2002;Wang et al, 2003). PA28␣␤ subunits are particularly abundant in immune tissues and are coordinately regulated by IFN␥ together with ␤1i, ␤2i, and ␤5i subunits of the immunoproteasome, ER peptide transporters (TAP1, TAP2), and MHC class I molecules (Frü h and Yang, 1999;Rechsteiner et al, 2000;Kloetzel and Ossendorp, 2004).…”

Section: Introductionmentioning

confidence: 99%

Global Organization and Function of Mammalian Cytosolic Proteasome Pools: Implications for PA28 and 19S Regulatory Complexes

Shibatani

Carlson

Larabee

et al. 2006

MBoC

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Proteolytic activity of the 20S proteasome is regulated by activators that govern substrate movement into and out of the catalytic chamber. However, the physiological relationship between activators, and hence the relative role of different proteasome species, remains poorly understood. To address this problem, we characterized the total pool of cytosolic proteasomes in intact and functional form using a single-step method that bypasses the need for antibodies, proteasome modification, or column purification. Two-dimensional Blue Native(BN)/SDS-PAGE and tandem mass spectrometry simultaneously identified six native proteasome populations in untreated cytosol: 20S, singly and doubly PA28-capped, singly 19S-capped, hybrid, and doubly 19S-capped proteasomes. All proteasome species were highly dynamic as evidenced by recruitment and exchange of regulatory caps. In particular, proteasome inhibition with MG132 markedly stimulated PA28 binding to exposed 20S ␣-subunits and generated doubly PA28-capped and hybrid proteasomes. PA28 recruitment virtually eliminated free 20S particles and was blocked by ATP depletion. Moreover, inhibited proteasomes remained stably associated with distinct cohorts of partially degraded fragments derived from cytosolic and ER substrates. These data establish a versatile platform for analyzing substrate-specific proteasome function and indicate that PA28 and 19S activators cooperatively regulate global protein turnover while functioning at different stages of the degradation cycle. INTRODUCTIONThe 20S proteasome is a cylindrical multicatalytic protease comprised of two stacked inner rings of proteolytically active ␤-subunits flanked by two outer rings of ␣-subunits (Lowe et al., 1995;Groll et al., 1997;Voges et al., 1999;Glickman and Ciechanover, 2002;Pickart and Cohen, 2004). In mammalian cells, proteasome activity is controlled by regulatory complexes (caps) that bind to the exposed ends of ␣-subunits and open the gate into and out of the catalytic chamber (Hoffman et al., 1992;DeMartino and Slaughter, 1999;Voges et al., 1999;Groll et al., 2000;Kloetzel and Ossendorp, 2004;Rechsteiner and Hill, 2005). One such cap, the 19S regulatory complex (PA700/RC), contains a hexameric ring of AAAATPases (base) and at least 12 additional subunits (lid) that recognize, unfold, and translocate polyubiquitinated proteins into the axial opening of the 20S core (Tanaka, 1998;Glickman et al., 1999;Strickland et al., 2000;Leggett et al., 2005;Liu et al., 2005). Two 19S RCs bind the 20S particle to form the doubly capped (30.3S) proteasomes (Yoshimura et al., 1993) that are generally believed to be the major species responsible for degrading polyubiquitinated proteins in the cell (Tanaka and Tsurumi, 1997;Voges et al., 1999;Wolf and Hilt, 2004). 19S RC binding to the 20S core particle requires ATP (Orino et al., 1991), and ATP hydrolysis transiently dissociates 20S and 19S particles, possibly to allow release of degradation products (Babbitt et al., 2005). Thus it has been proposed that the degradation cycle i...

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“…ABPs targeting serine proteases (81,82), cysteine proteases (85)(86)(87)(88), threonine proteases (89,90), aspartyl proteases (91), and metalloproteases (92,93) have been developed. Metallo-and aspartic proteases pose difficulty in the design and application of ABPs due to the absence of a covalent acyl intermediate in the catalytic mechanism.…”

Section: Activity-based Probesmentioning

confidence: 99%

Metadegradomics

Doucet

Butler

Rodrı́guez

et al. 2008

Molecular & Cellular Proteomics

138

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Post-translational modifications enable extra layers of control of the proteome, and perhaps the most important is proteolysis, a major irreversible modification affecting every protein. The intersection of the protease web with a proteome sculpts that proteome, dynamically modifying its state and function. Protease expression is distorted in cancer, so perturbing signaling pathways and the secretome of the tumor and reactive stromal cells. Indeed many cancer biomarkers are stable proteolytic fragments. It is crucial to determine which proteases contribute to the pathology versus their roles in homeostasis and in mitigating cancer. Thus the full substrate repertoire of a protease, termed the substrate degradome, must be deciphered to define protease function and to identify drug targets. Degradomics has been used to identify many substrates of matrix metalloproteinases that are important proteases in cancer. Here we review recent degradomics technologies that allow for the broadly applicable identification and quantification of proteases (the protease degradome) and their activity state, substrates, and interactors. Quantitative proteomics using stable isotope labeling, such as ICAT, isobaric tags for relative and absolute quantification (iTRAQ), and stable isotope labeling by amino acids in cell culture (SILAC), can reveal protease substrates by taking advantage of the natural compartmentalization of membrane proteins that are shed into the extracellular space. Identifying the actual cleavage sites in a complex proteome relies on positional proteomics and utilizes selection strategies to enrich for proteasegenerated neo-N termini of proteins. In so doing, important functional information is generated. Finally protease substrates and interactors can be identified by interactomics based on affinity purification of protease complexes using exosite scanning and inactive catalytic domain capture strategies followed by mass spectrometry analysis. At the global level, the N terminome analysis of whole communities of proteases in tissues and organs in vivo provides a full scale understanding of the protease web and the web-sculpted proteome, so defining metadegradomics. Molecular & Cellular Proteomics 7: 1925-1951, 2008.

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Biochemical Analysis of the 20 S Proteasome of Trypanosoma brucei

Cited by 44 publications

References 41 publications

Distinct Specificities of Mycobacterium tuberculosis and Mammalian Proteasomes for N-Acetyl Tripeptide Substrates

Distinct Specificities of Mycobacterium tuberculosis and Mammalian Proteasomes for N-Acetyl Tripeptide Substrates

Global Organization and Function of Mammalian Cytosolic Proteasome Pools: Implications for PA28 and 19S Regulatory Complexes

Metadegradomics

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