The 2004 Nobel Prize in chemistry for the discovery of protein ubiquitination has led to the recognition of cellular proteolysis as a central area of research in biology. Eukaryotic proteins targeted for degradation by this pathway are first 'tagged' by multimers of a protein known as ubiquitin and are later proteolyzed by a giant enzyme known as the proteasome. This article recounts the key observations that led to the discovery of ubiquitin-proteasome system (UPS). In addition, different aspects of proteasome biology are highlighted. Finally, some key roles of the UPS in different areas of biology and the use of inhibitors of this pathway as possible drug targets are discussed.

PepN and its homologues are involved in the ATP-independent steps (downstream processing) during cytosolic protein degradation. To obtain insights into the contribution of PepN to the peptidase activity in Escherichia coli, the hydrolysis of a selection of endopeptidase and exopeptidase substrates was studied in extracts of wild-type strains and two pepN mutants, 9218 and DH5aDpepN. Hydrolysis of three of the seven endopeptidase substrates tested was reduced in both pepN mutants. Similar studies revealed that hydrolysis of 10 of 14 exopeptidase substrates studied was greatly reduced in both pepN mutants. This decreased ability to cleave these substrates is pepN-specific as there is no reduction in the ability to hydrolyse exopeptidase substrates in E. coli mutants lacking other peptidases, pepA, pepB or pepE. PepN overexpression complemented the hydrolysis of the affected exopeptidase substrates. These results suggest that PepN is responsible for the majority of aminopeptidase activity in E. coli. Further in vitro studies with purified PepN revealed a preference to cleave basic and small amino acids as aminopeptidase substrates. Kinetic characterization revealed the aminopeptidase cleavage preference of E. coli PepN to be Arg>Ala>Lys>Gly. Finally, it was shown that PepN is a negative regulator of the sodium-salicylate-induced stress in E. coli, demonstrating a physiological role for this aminoendopeptidase under some stress conditions. INTRODUCTIONThe mechanisms involved in cytosolic protein degradation are central to regulating various aspects of cell biology, including cell cycle, disease progression, transcriptional regulation, antigen processing, stress responses, etc. Proteins targeted for degradation are unfolded and cleaved in an ATP-dependent manner to release large peptides, ranging from 3 to 25 amino acids (Maurizi, 1987;Coux et al., 1996;Gottesman, 1996;Kisselev et al., 1999;Zwickl et al., 2000). These are further trimmed, cleaved and/or degraded by several endopeptidases, tri-and di-peptidyl peptidases (Tamura, T. et al., 1996; Fukasawa et al., 1998;Osmulski & Gaczynska, 1998;Tamura, N. et al., 1998;Geier et al., 1999;Wang et al., 2000), aminopeptidases and carboxypeptidases (Conlin & Miller, 1995; Gonzales & RobertBaudouy, 1996;Chandu & Nandi, 2002;Franzetti et al., 2002) in an ATP-independent manner, also known as downstream processing. This general scheme of cytosolic protein degradation is conserved in all organisms, although the enzymes involved are distinct in different organisms. Most of the enzymes and their homologues that are upstream in the proteolysis pyramid, i.e. endopeptidases (e.g. Tricorn, tripeptidyl peptidase II, bleomycin hydrolase, thimet oligopeptidase), are present in selected organisms. However, the enzymes and their homologues involved in the latter steps of downstream processing, for example, leucine aminopeptidase and puromycin-sensitive aminopeptidase, are present in most organisms (Chandu & Nandi, 2002). Also, prokaryotes display greater redundancy than eukaryotes in the ...

Presenilins (PS) provide the catalytic activity for ␥-secretase, which cleaves physiologically relevant substrates including Notch, ErbB4, and APP. Recent genetic studies indicated that the contribution of PS1 to mouse development includes ␥-secretase-independent functions that cannot be easily explained by any of the demonstrated or hypothesized functions of this protein. To begin a nonbiased analysis of PS1 activity unencumbered by the dominant effect stemming from loss of Notch function, we characterized PS functions in the early land plant Physcomitrella patens, which lacks Notch, ErbB4, and APP. Removal of P. patens PS resulted in phenotypic abnormalities. Further assays performed to delineate the defective pathways in PS-deficient P. patens implicated improper function of the cytoskeletal network. Importantly, this characterization of a nonmetazoan PS uncovered a previously undescribed, evolutionarily conserved function (human PS1 can rescue the growth and light responses) that is ␥-secretase-independent (mutants with substitutions of the catalytic aspartyl residues retain the activity). Introduction of PpPS into PS-deficient mouse embryonic fibroblasts rescues normal growth rates, demonstrating that at least some metazoan functions of PS are evolutionarily conserved.

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...

The general pathway involving adenosine triphosphate (ATP)-dependent proteases and ATP-independent peptidases during cytosolic protein degradation is conserved, with differences in the enzymes utilized, in organisms from different kingdoms. Lon and caseinolytic protease (Clp) are key enzymes responsible for the ATP-dependent degradation of cytosolic proteins in Escherichia coli. Orthologs of E. coli Lon and Clp were searched for, followed by multiple sequence alignment of active site residues, in genomes from seventeen organisms, including representatives from eubacteria, archaea, and eukaryotes. Lon orthologs, unlike ClpP and ClpQ, are present in most organisms studied. The roles of these proteases as essential enzymes and in the virulence of some organisms are discussed.