Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway

Falnes, Pål Ø.; Olsnes, Sjur

doi:10.1093/emboj/17.2.615

Cited by 34 publications

(38 citation statements)

References 62 publications

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“…However, the only known functions of LLO and the phospholipases are required in vacuoles, where presumably they would be protected from degradation via the N-end rule pathway. Recently, in a study similar to the one presented here, it was shown that diphtheria toxin is degraded by the N-end rule pathway of eukaryotic cells (Falnes and Olsnes, 1998). Moreover, it was demonstrated that the half-life of this toxin, whose site of action is in the host cell cytosol, corresponds to its degree of toxicity in Vero cells.…”

Section: Discussionsupporting

confidence: 64%

“…Moreover, it was demonstrated that the half-life of this toxin, whose site of action is in the host cell cytosol, corresponds to its degree of toxicity in Vero cells. The results of the present study and that of Falnes and Olsnes (1998) suggest that there is a general relationship between subcellular location, half-life and function of virulenceassociated proteins, a possibility that warrants further investigation. In summary, we have shown that the N-terminus of an intracellular bacterial protein can in¯uence its degradation rate and, consequently, its function.…”

Section: Discussionsupporting

confidence: 51%

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Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway

1999

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SummaryUpon infection of mammalian cells, Listeria monocytogenes lyses the phagosome and enters the cytosol, where it secretes proteins necessary for its intracellular growth cycle. Consequently, bacterial proteins exposed to the cytosol are potential targets for degradation by host cytosolic proteases. One pathway for degradation of host cytosolic proteins, the N-end rule pathway, involves recognition of the N-terminal amino acid and is mediated by the proteasome. However, very few natural N-end rule substrates have been identi®ed. We have examined the L. monocytogenes ActA protein as a potential target for this pathway. ActA is an essential determinant of L. monocytogenes pathogenesis that is required to induce actin-based motility and cell-to-cell spread. We show that the half-life of a secreted form of ActA can be altered in the mammalian cytosol by changing the N-terminal amino acid. Moreover, the introduction of a destabilizing N-terminus into the functional, surface-bound form of ActA results in a small-plaque phenotype in L2 cells, which is partially reversible by an inhibitor of the proteasome. These results indicate that the L. monocytogenes ActA protein is a natural N-end rule substrate, and that optimal function of ActA in mediating cell-to-cell spread is dependent upon its intracellular turnover rate.

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Section: Discussionsupporting

confidence: 64%

Section: Discussionsupporting

confidence: 51%

Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway

1999

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“…Indeed, protease inhibitors are known to increase the cytotoxicity of other protein cytotoxins (57). Similarly, the addition of proteolytic degradation signals can decrease protein cytotoxicity (58). Finally, it is noteworthy that protein toxins such as diphtheria toxin, enterotoxin, and abrin II have T m values near that of RNase A (59 -61).…”

Section: Discussionmentioning

confidence: 99%

Conformational Stability Is a Determinant of Ribonuclease A Cytotoxicity

Klink¹,

Raines²

2000

Journal of Biological Chemistry

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Onconase™, a homolog of bovine pancreatic ribonuclease A (RNase A) with high conformational stability, is cytotoxic and has efficacy as a cancer chemotherapeutic agent. Unlike wild-type RNase A, the G88R variant is toxic to cancer cells. Here, variants in which disulfide bonds were removed from or added to G88R RNase A were used to probe the relationship between conformational stability and cytotoxicity in a methodical manner. The conformational stability of the C40A/G88R/C95A and C65A/C72A/G88R variants is less than that of G88R RNase A. In contrast, a new disulfide bond that links the N and C termini (residues 4 and 118) increases the conformational stability of G88R RNase A and C65A/C72A/ G88R RNase A. These changes have little effect on the ribonucleolytic activity of the enzyme or on its ability to evade the cytosolic ribonuclease inhibitor protein. The changes do, however, have a substantial effect on toxicity toward human erythroleukemia cells. Specifically, conformational stability correlates directly with cytotoxicity as well as with resistance to proteolysis. These data indicate that conformational stability is a key determinant of RNase A cytotoxicity and suggest that cytotoxicity relies on avoiding proteolysis. This finding suggests a means to produce new cancer chemotherapeutic agents based on mammalian ribonucleases. The free energy difference between the native and unfolded states of a protein is small-typically 5-15 kcal/mol (1, 2). In their native state, many proteins are less susceptible to proteolytic degradation than when unfolded (3). In the unfolded state, the steric protection of peptide bonds provided by the compact native state is lost (4). For example, bovine pancreatic ribonuclease A (RNase A) 1 (EC 3.1.27.5 (5, 6)) is degraded more readily by proteases in the presence of denaturants or elevated temperatures (7-10) and less readily when glycosylated (11).The rates of intracellular protein turnover vary by 10 3 -fold (12). Apparently, some proteins are better able to thwart the proteolytic machinery (13,14). These proteins remain intact and retain activity longer within the cell. A correlation between the conformational stability of a protein and its catabolism was reported over 20 years ago (15). Since then, studies using unrelated proteins have either supported (16) or contradicted (14, 17) this correlation. Drawing conclusions from these studies is problematic because the proteins were divergent in characteristics that have been implicated in metabolic turnover. In contrast, variants of a single protein, the N-terminal domain of the repressor protein from bacteriophage , have been used to demonstrate a definite link between conformational stability and metabolic turnover (18). More recently, the conformational stability of bovine pancreatic trypsin inhibitor variants was shown to correlate with the yield of intact protein produced in a heterologous system (19).RNase A homologs elicit diverse biological activities, including specific toxicity to cancer cells (20,21). Onconase™, which ...

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“…The delay in apoptosis to 48-60 h corresponds with the time required for toxin internalization, translocation and EF2 inactivation. 30 Mutation of the p53 gene and loss of its function is frequently observed in acute phase CML. 31 Mutant p53 cells have poor apoptosis induction by conventional cytotoxic drugs.…”

Section: Discussionmentioning

confidence: 99%

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias

et al. 2000

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Leukemic blasts from patients with acute phase chronic myeloid leukemic and refractory acute myeloid leukemia are highly resistant to a number of cytotoxic drugs. To overcome multi-drug resistance, we engineered a diphtheria fusion protein by fusing human interleukin-3 (IL3) to a truncated form of diphtheria toxin (DT) with a (G 4 S) 2 linker (L), expressed and purified the recombinant protein, and tested the cytotoxicity of the DTLIL3 molecule on human leukemias and normal progenitors. The DTLIL3 construct was more cytotoxic to interleukin-3 receptor (IL3R) bearing human myeloid leukemia cell lines than receptor-negative cell lines based on assays of cytotoxicity using thymidine incorporation, growth in semi-solid medium and induction of apoptosis. Exposure of mononuclear cells to 680 pM DTLIL3 for 48 h in culture reduced the number of cells capable of forming colonies in semi-solid medium (colony-forming units leukemia) у10-fold in 4/11 (36%) patients with myeloid acute phase chronic myeloid leukemia (CML) and 3/9 (33%) patients with acute myeloid leukemia (AML). Normal myeloid progenitors (colony-forming unit granulocytemacrophage) from five different donors treated and assayed under identical conditions showed intermediate sensitivity with three-to five-fold reductions in colonies. The sensitivity to DTLIL3 of leukemic progenitors from a number of acute phase CML patients suggests that this agent could have therapeutic potential for some patients with this disease. Leukemia (2000) 14, 576-585.

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Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway

Cited by 34 publications

References 62 publications

Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway

Stability of the Listeria monocytogenes ActA protein in mammalian cells is regulated by the N-end rule pathway

Conformational Stability Is a Determinant of Ribonuclease A Cytotoxicity

Diphtheria toxin fused to human interleukin-3 is toxic to blasts from patients with myeloid leukemias

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