The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus

Tamura, Tomohiro; Nagy, István; Lupas, Andrei N.; Lottspeich, Friedrich; Cejka, Zdenka; Schoofs, Geert; Tanaka, Keiji; Mot, René De; Baumeister, Wolfgang

doi:10.1016/s0960-9822(95)00153-9

Cited by 179 publications

(155 citation statements)

References 42 publications

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“…Recently it has become clear that bacteria encoding a proteasome [9][10][11], like for example Mycobacterium tuberculosis (Mtb), use a similar tagging system termed pupylation [12][13][14]. In this process, the prokaryotic ubiquitin-like protein (Pup) is conjugated to substrate-lysines via its deamidated C-terminal GGQ-motif [12,14].…”

Section: Introductionmentioning

confidence: 99%

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

et al. 2009

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Edited by Miguel De la RosaKeywords: Prokaryotic ubiquitin-like protein Mpa ARC Proteasome NMR Mycobacterium tuberculosis a b s t r a c tThe mycobacterial ubiquitin-like protein Pup is coupled to proteins, thereby rendering them as substrates for proteasome-mediated degradation. The Pup-tagged proteins are recruited by the proteasomal ATPase Mpa (also called ARC). Using a combination of biochemical and NMR methods, we characterize the structural determinants of Pup and its interaction with Mpa, demonstrating that Pup adopts a range of extended conformations with a short helical stretch in its C-terminal portion. We show that the N-terminal coiled-coil domain of Mpa makes extensive contacts along the central region of Pup leaving its N-terminus unconstrained and available for other functional interactions. Structured summary:MINT-7262427: pup (uniprotkb:B6DAC1) binds (MI:0407) to mpa (uniprotkb:Q0G9Y7) by pull down (MI:0096) MINT-7262440: mpa (uniprotkb:Q0G9Y7) and pup (uniprotkb:B6DAC1) bind (MI:0407) by isothermal titration calorimetry (MI:0065)

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Section: Introductionmentioning

confidence: 99%

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

et al. 2009

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“…Although the E. coli genome does not encode "true" 20 S proteasome subunits (28), an LMW Suc-LLVY-AMC hydrolyzing activity was detected. There are reports in two bacteria, Rhodococcus (24) and Frankia (25), and mouse liver (27) where an LMW Suc-LLVY-AMC-cleaving peptidase was also detected. However, the enzyme(s) responsible for this activity have not been identified.…”

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confidence: 98%

“…Suc-LLVY-AMC, a chymotryptic peptidase substrate, is cleaved by eubacterial (24,25), archaeal (26), and mammalian (7,27) 20 S proteasomes. Although the E. coli genome does not encode "true" 20 S proteasome subunits (28), an LMW Suc-LLVY-AMC hydrolyzing activity was detected.…”

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confidence: 99%

PepN, the Major Suc-LLVY-AMC-hydrolyzing Enzyme inEscherichia coli, Displays Functional Similarity with Downstream Processing Enzymes in Archaea and Eukarya

Chandu

Kumar

Nandi

2003

Journal of Biological Chemistry

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Succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin (Suc-LLVY-AMC), a fluorogenic endopeptidase substrate, is used to detect 20 S proteasomal activity from Archaea to mammals. An o-phenanthroline-sensitive Suc-LLVY-AMC hydrolyzing activity was detected in Escherichia coli although it lacks 20 S proteasomes. We identified PepN, previously characterized as the sole alanine aminopeptidase in E. coli, to be responsible for the hydrolysis of Suc-LLVY-AMC. PepN is an aminoendopeptidase. First, extracts from an ethyl methanesulfonate-derived PepN mutant, 9218, did not cleave Suc-LLVY-AMC and L-Ala-para-nitroanilide (pNA). Second, biochemically purified PepN cleaves a wide variety of both aminopeptidase and endopeptidase substrates, and L-Ala-pNA is cleaved more efficiently than other substrates. Studies with bestatin, an aminopeptidasespecific inhibitor, suggest differences in the mechanisms of cleavage of aminopeptidase and endopeptidase substrates. Third, PepN hydrolyzes whole proteins, casein and albumin. Finally, an E. coli strain with a targeted deletion in PepN also lacks the ability to cleave Suc-LLVY-AMC and L-Ala-pNA, and expression of wild type PepN in this mutant rescues both activities. In addition, we identified a low molecular weight Suc-LLVY-AMC-cleaving peptidase in Mycobacterium smegmatis, a eubacteria harboring 20 S proteasomes, to be an aminopeptidase homologous to E. coli PepN, by mass spectrometry analysis. "Sequence-based homologues" of PepN include well characterized aminopeptidases, e.g. Tricorn interacting factors F2 and F3 in Archaea and puromycin-sensitive aminopeptidase in mammals. However, our results suggest that eubacterial PepN and its homologues displaying aminoendopeptidase activities may be "functionally similar" to enzymes important in downstream processing of proteins in the cytosol: Tricorn-F1-F2-F3 complex in Archaea and TPPII/Multicorn in eukaryotes.Dynamic changes in the proteome of a cell depend on rates of protein synthesis and their degradation. The past few decades have witnessed enormous strides in identifying the molecules and understanding the mechanisms involved in intracellular protein degradation. There is increasing evidence of the involvement of protein degradation in diverse biological activities, e.g. cell cycle progression, transcriptional activation, antigen processing, disease progression, etc. (1-5).Broadly, cytosolic protein degradation is categorized into four steps. (i) Proteins targeted for degradation are initially unfolded into polypeptides by ATP-dependent proteases belonging to the Lon/Clp family in bacteria or 26 S proteasomes in higher organisms (1-5). (ii) These enzymes also make the initial endoproteolytic "cuts" in the polypeptide. Interestingly, in both Escherichia coli (6) and higher organisms (7) the average length of peptides released by these enzymes range from 3 to 25 amino acids. (iii) These longer peptides are trimmed into smaller peptides (less than 10 amino acids) by the action of endopeptidases (8 -11), tripeptidyl-and dipeptidylpeptidases...

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“…However, 20S proteasomes were recently discovered in the actinomycete Rhodococcus (12), and in the Escherichia coli genome sequencing project, a novel heat-shock locus (hslVU) was discovered that encodes a 19-kDa protein (HslV) (13), whose sequence is similar to 3-type proteasome subunits. This discovery of proteasome-related genes was surprising, because several groups had failed to observe a structure in E. coli resembling the proteasome or proteins resembling ubiquitin.…”

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“…Although the HslVU protease degraded Z-Gly-Gly-Leu-AMC rapidly, it hydrolyzed Suc-Ala-Ala-Phe-AMC and SucLeu-Leu-Val-Tyr-AMC very slowly, which are rapidly hydrolyzed by chymotrypsin and proteasomes from Thermoplasma (11,25) or Rhodococcus (12) (Table 4). In addition, ATP activated Z-Gly-Gly-Leu-AMC hydrolysis up to 150-fold and stimulated cleavage of Suc-Ala-Ala-Phe-AMC and Suc-Leu-LeuVal-Tyr-AMC only 3-to 5-fold (data not shown).…”

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HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome.

Rohrwild

Coux

Huang

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

216

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We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hsIVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome (3 subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coi expressing heat-shock Proteasomes are multicatalytic proteolytic complexes present in both the nucleus and cytosol of eukaryotic cells (1). The 26S form of the proteasome catalyzes the degradation of ubiquitinconjugated proteins (2-5), and thus it plays a key role in many cellular processes, including progression through the cell cycle (6, 7), removal of abnormal proteins, and antigen presentation (8). The proteolytic core of the 26S complex is the 20S (700 kDa) proteasome particle, which consists of four sevenmembered rings. The subunits of the 20S proteasome fall into two families (9, 10): the a-type forms the two outer rings, and P-type, which contain the active sites, forms the two inner rings of the complex.Proteasomes were thought to exist exclusively in eukaryotes and certain archaebacteria (11). However, 20S proteasomes were recently discovered in the actinomycete Rhodococcus (12), and in the Escherichia coli genome sequencing project, a novel heat-shock locus (hslVU) was discovered that encodes a 19-kDa protein (HslV) (13), whose sequence is similar to 3-type proteasome subunits. This discovery of proteasome-related genes was surprising, because several groups had failed to observe a structure in E. coli resembling the proteasome or proteins resembling ubiquitin. The hslV gene is cotranscribed with the adjacent hslU gene, which codes for a 50-kDa protein containing one ATP/GTP binding motif (13 For the expression of glutathione S-transferase (GST)-fusion proteins, the hslVand hslU genes were PCR amplified separately using A phage 18-126 DNA bearing the hslVU operon, kindly provided by F. Blattner (University of Wisconsin-Madison), and cloned into the vector pGEX-2T (Pharmacia). Vector pV106 (GST-HslV) and pU206 (GST-HslU) were electroporated into E. coli C600 cells. GST-fusion proteins were purified from strains C106 and C206 using the GST Purification Module (Pharmacia). To obtain antibodies, purified GST-HslV and GST-HslU proteins were injected into rabbits. Polyclonal anti-HslV and antiHslU antibodies were then affinity purified using the GST-fusions as ligands, and depleted of anti-GST antibodies using a GST column.Abbreviations: hsl, heat-shock locus; GST, glutathone S-transferase. tTo whom reprint requests should be addressed.5808

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The first characterization of a eubacterial proteasome: the 20S complex of Rhodococcus

Abstract: The recent description of the first eubacterial ubiquitin, and our discovery of a eubacterial proteasome show that the ubiquitin pathway of protein degradation is ancestral and common to all forms of life.

Cited by 179 publications

References 42 publications

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

A distinct structural region of the prokaryotic ubiquitin‐like protein (Pup) is recognized by the N‐terminal domain of the proteasomal ATPase Mpa

PepN, the Major Suc-LLVY-AMC-hydrolyzing Enzyme inEscherichia coli, Displays Functional Similarity with Downstream Processing Enzymes in Archaea and Eukarya

HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome.

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