The heat-stable polypeptide ATP-dependent proteolysis factor 1 (APF-1) of the reticulocyte proteolytic system forms covalent compounds with proteins in an ATP-requiring reaction. APF-1 and lysozyme, a good substrate for ATP-dependent proteolysis, form multiple conjugates, as was shown by comigration of label from each upon gel electrophoresis.Multiple bands were also seen with other substrates of the ATP-dependent proteolytic system, such as globin or a-lactalbumin. Analysis of the ratio of APF-1 to lysozyme radioactivities and of the molecular weights of the bands indicated that they consist of increasing numbers of the APF-1 polypeptide bound to one molecule of lysozyme. The covalent linkage is probably of an isopeptide nature, because it is stable to hydroxylamine and alkali, and polylysine is able to give conjugates of APF-1. Removal of ATP after formation of the 125I-labeled APF-1 conjugates with endogenous proteins caused the regeneration of APF-1, indicating presence of an amidase. This reaction is thought to compete with proteases that may act on APF-1-protein conjugates, especially those containing several APF-1 ligands. A sequence of reactions in which the linkage of APF-1 to the substrate is followed by the proteolytic breakdown of the substrate is proposed to explain the role of ATP.Since the discovery of the energy requirement of protein breakdown by Simpson in 1953 (1), this feature has been found to be universal in a variety of biological systems (for review, see ref.2). Only recently have cell-free systems in which protein breakdown is stimulated by ATP been delineated (3-6).We have resolved the ATP-dependent proteolytic system from reticulocytes into several components (7,8). In the preceding paper (9) it was found that the heat-stable polypeptide of this system, ATP-dependent proteolysis factor 1 (APF-1), is bound to reticulocyte proteins in an ATP-specific reaction. We now show that this is due to the formation of a covalent, probably amide, linkage between the protein substrate and one or more APF-1 molecules. The role of this reaction in protein breakdown is considered. METHODS Preparation of Radiolabeled Proteins and EnzymeFractions. The purification and radiolabeling of APF-1 from rabbit reticulocytes were described in the previous article (9). Crystalline hen egg white lysozyme (Worthington) and purified bovine a-lactalbumin (a generous gift of W. Klee) were labeled with Na'25I by a method similar to that described for the radioiodination of APF-1 (9). When used in unlabeled form these proteins were treated in the same way without isotope. Globin labeled with [3H]leucine was prepared as described (7). Fraction 11 (0.5 M KCI eluate from DEAE-cellulose) was prepared from ATP-depleted rabbit reticulocytes (7). Poly(L-lysine) was obtained from Miles-Yeda (Mr 1500-8000). (vol/vol) glycerol, 0.5% NaDodSO4, and 0.5% 2-mercaptoethanol. The samples were electrophoresed on NaDodSO4/polyacrylamide slab gels (13 X 13 X 0.12 cm) with the system of Laemmli (10).Unless otherwise stated, 10-14...

The heat-stable polypeptide (APF-1) required for ATP-dependent proteolysis in reticulocytes enters into high molecular weight conjugates upon incubation with the fraction of reticulocytes that is retained by DEAE-cellulose. Conjugate formation requires ATP and Mg2+ and is inhibited by N-ethylmaleimide. UTP and GTP are inactive. These properties are identical to those of ATP-dependent protein breakdown in the same system, suggesting that the conjugates are intermediates in this process. The APF-1 conjugates are stable in sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Sephadex C-75 isolation and are resistant to mild acid, alkali, heat denaturation, and reduction; the conjugates are therefore covalent.Although most cellular proteins turn over rapidly, the enzymic reactions of protein degradation have not yet been identified. A basic feature of intracellular protein breakdown is its absolute requirement for cellular energy. Inhibitors of ATP production inhibit the degradation of liver proteins (1, 2), of tyrosine aminotransferase in hepatoma cells (3), of normal proteins in bacteria (4, 5) and cultured cells (6, 7), and of abnormal proteins in Escherichia coli (8, 9) and reticulocytes (10, 11). Recently several reports have shown an ATP requirement for protein breakdown in cell-free systems. ATP stimulates the degradation of abnormal proteins in crude soluble extracts of reticulocytes (10, 11) and E. coli (12), and a requirement for ATP has been found in the cleavage of bacteriophage X repressor in vitro (13,14).We have shown that the ATP-dependent proteolytic system from rabbit reticulocytes is composed of several required components. A heat-stable polypeptide of a relatively small size, (Mr -8000) designated ATP-dependent proteolysis factor 1 (APF-1), has been resolved (15). APF-1 has no proteolytic activity itself but stimulates ATP-dependent protein breakdown by a crude protein fraction eluted from DEAE-cellulose, fraction 11 (15). Fraction II has been resolved into two subfractions, both required for protein breakdown by the ATP-APF-1 system (16).To gain a better insight into the roles of the different factors and of ATP in protein breakdown, we have now purified APF-1, radiolabeled it, and observed its association with other reticulocyte components. ATP is required for binding of APF-1 to reticulocyte proteins and the binding seems to be covalent in nature. METHODS Preparation of Enzyme Fractions and Purification of APF-1. Lysates from ATP-depleted rabbit reticulocytes were prepared and separated on DEAE-cellulose into fraction I (unadsorbed material) and fraction 11 (0.5 M KCI eluate), as described earlier (15, 16). Fraction I was subjected to heat treatment (90'C, 20 min) and gel filtration on a column (1.5 X 90 cm) of Sephadex G-75, as described (15). Then 10 mg of this material was adsorbed onto a column (1.5 X 30 cm) of CM-Sephadex equilibrated with 10 mM potassium phosphate and was eluted with a 0-150 mM linear gradient of KCl. The active peak, designated APF-1, eluted at about 6...

ISG15 is a 15-kDa protein of unique primary amino acid sequence, which is transcriptionally regulated by interferon (IFN) cx and IFN-f3. Because it is synthesized in many cell types and secreted from human monocytes and lymphocytes, we postulated that ISG15 might act to modulate immune cell function. ISG15 stimulated B-depleted lymphocyte proliferation in a dose-dependent manner with significant proliferation induced by amounts of ISG15 as low as 1 ng/ml (58 pM Although molecular mechanisms underlying biological responses to interferons (IFNs) are only partially dissected, it is thought that they are mediated by the regulated synthesis of induced proteins (1, 2). One of these IFN-induced gene products is ISG15 (3,4). ISG15 is a 15-kDa protein that is transcriptionally regulated by IFN-a or B (5,6). ISG15 is synthesized in mammalian cells as a 17-kDa precursor (pre-ISG15) that is processed by a cellular converting enzyme, cleaving the 8 carboxyl-terminal aa to yield the 157-aa mature ISG15 (4, 7), which is secreted from monocytes and lymphocytes (8). Both native and recombinant ISG15 induce the synthesis and secretion of IFN-'y from B-depleted lymphocytes (9).Based on its cytokine-like properties, it was hypothesized that ISG15 modulates immune effector cell activation and function. To evaluate this, effects on peripheral blood lymphocytes were assessed by [3Hlthymidine incorporation, immunophenotyping, and cytolytic assays. We demonstrate that ISG15 induced production of IFN-,y from T cells, augmented the proliferation of natural killer (NK) cells, and inducedThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.non-major histocompatibility complex-restricted cytolysis of tumor cell targets by NK-derived lymphokine-activated killer (LAK) cells in the absence of detectable levels of interleukin (IL) 2 or IL-12.MATERIALS AND METHODS Materials. B-depleted peripheral blood lymphocytes (PBLs) were purified by passage over a nylon wool column (9). By flow cytometry, this population was 80-85% CD3+, 5-8% CD16+, <2% CD14+, and <2% CD19+. Human recombinant IL-2 (2 x 107 units/mg) was from Hoffmann-LaRoche. Human recombinant IFN-y (2 x 107 units/mg) was from Biogen. A polyclonal antiserum to homogenous ISG15, purified from cytoplasms of IFN-,B-treated Daudi cells, was raised in a New Zealand White rabbit (7). Antigenic specificities of monoclonal antibodies used for flow cytometry included CD3 (Leu 4), CD14 (Leu M3), CD16 (Leu lla), CD19 (Leu 12), and CD56 (NKH-1) (Becton Dickinson).Expression and Purification of ISG15 and pre-ISG15. Human 157-aa ISG15 and the 165-aa pre-ISG15 were expressed and purified from Escherichia coli BL21(DE3) (10, 11). Endotoxin levels were <0.03 endotoxin units (EU)/,tg as measured by limulus amoebocyte lysate assay (sensitivity, 0.01 EU/ml). Heat-denatured ISG15 was ISG15 boiled for 20 min.Functional Assays. Proliferation ([3H]thy...

The covalent attachment of the polypeptide ubiquitin to proteins marks them for degradation by the ubiquitin/26S proteasome-dependent degradation pathway. This pathway functions in regulating many fundamental processes required for cell viability. Phylogenetic analysis of ubiquitin sequences reveals greater variability among lower eukaryotes and defines essential residues, many of which are conserved among the three ubiquitin-like proteins known to undergo parallel ligation pathways. The hierarchical design of the ubiquitin conjugation mechanism provides great flexibility for the divergent evolution of new functions mediated by this posttranslational modification. Within this hierarchy, a single ubiquitin-activating enzyme provides charged intermediates to multiple targeting pathways defined by cognate ubiquitin carrier protein (E2)/ligase (E3) pairs. Sequence analysis of E2 isozymes shows that the E2 superfamily is composed of distinct function-specific families. The apparent lack of E2/E3 specificity suggested in the literature results from the presence of multiple isozymes within many E2 families and erroneous family assignments based on incomplete data sets. Other apparent inconsistencies are explained by interfamily sequence relationships among some E2 isoforms.

IFN-stimulatory gene factor 15 (ISG15) is a ubiquitin-like protein, which is conjugated to many cellular proteins. However, its role in protein degradation is unclear. Here, we show that ISG15 is highly elevated and extensively conjugated to cellular proteins in many tumors and tumor cell lines. The increased levels of ISG15 in tumor cells were found to be associated with decreased levels of polyubiquitinated proteins. Specific knockdown of ISG15 expression using ISG15-specific small interfering RNA (siRNA) was shown to increase the levels of polyubiquitinated proteins, suggesting an antagonistic role of ISG15 in regulating ubiquitin-mediated protein turnover. Moreover, siRNA-mediated down-regulation of the major E2 for ISG15 (UbcH8), which blocked the formation of ISG15 protein conjugates, also increased the levels of polyubiquitinated proteins. Together, our results suggest that the ISG15 pathway, which is deregulated during tumorigenesis, negatively regulates the ubiquitin/proteasome pathway by interfering with protein polyubiquitination/ degradation. (Cancer Res 2006; 66(2): 921-8)