Kinetic Analysis of the Conjugation of Ubiquitin to Picornavirus 3C Proteases Catalyzed by the Mammalian Ubiquitin-protein Ligase E3α

T Glen Lawson,Molly E Sweep,Peter E Schlax,Richard N Bohnsack,Arthur L Haas

doi:10.1074/jbc.m102659200

T Glen Lawson, Molly E Sweep + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m102659200

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Abstract

The 3C proteases of the encephalomyocarditis virus and the hepatitis A virus are both type III substrates for the mammalian ubiquitin-protein ligase E3alpha. The conjugation of ubiquitin to these proteins requires internal ten-amino acid-long protein destruction signal sequences. To evaluate how these destruction signals modulate interactions that must occur between E3alpha and the 3C proteases, we have kinetically analyzed the formation of ubiquitin-3C protease conjugates in a reconstituted system of purified E1, HsUbc2b/E2(14Kb), and human E3alpha. Our measurements show that the encephalomyocarditis virus 3C protease is ubiquitinated in this system with K(m) = 42 +/- 11 microm and V(max) = 0.051 +/- 0.01 pmol/min whereas the parameters for the ubiquitination of the hepatitis A virus 3C protease are K(m) = 20 +/- 5 microm and V(max) = 0.018 +/- 0.003 pmol/min. Mutations in the destruction signal sequences resulted in changes in the rate at which E3alpha conjugates ubiquitin to the altered 3C protease proteins. The K(m) and V(max) values for these reactions change proportionally in the same direction. These results suggest differences in rates of conjugation of ubiquitin to 3C proteases are primarily a k(cat) effect. Replacing specific encephalomyocarditis virus 3C protease lysine residues with arginine residues was found to increase, rather than decrease, the rate of ubiquitin conjugation, and the K(m) and V(max) values for these reactions are both higher than for the wild type protein. The ability of E3alpha to catalyze the conjugation of ubiquitin to both 3C proteases was found to be inhibited by lysylalanine and phenylalanylalanine, demonstrating that the same sites on E3alpha that bind destabilizing N-terminal amino acids in type I and II substrates also interact with the 3C proteases.

Highlights

The selection of proteins for destruction by the ubiquitin 26 S/proteasome pathway depends upon specific interactions that occur between the targeted substrates and enzymes involved in the formation of the ubiquitin-target protein conjugates
We employed a biochemically defined, reconstituted N-end rule ubiquitin ligation system, comprised of affinity-purified human E1 and E3␣ and recombinant human HsUbc2b, to quantitatively evaluate the targeting of the encephalomyocarditis virus (EMCV) and hepatitis A virus (HAV) 3C proteases for ubiquitin attachment and to assess the effect disrupting their respective destruction signal sequences has on their selection as N-end rule pathway substrates
Because our goal in this study was to attempt to detect potentially subtle differences in the kinetics with which related substrate proteins are ubiquitinated by the same ubiquitin-protein ligase, we preferred to avoid artifacts that might result from the use of ubiquitin mutants or derivatives that do not support polyubiquitin chain synthesis

Summary

The abbreviations used are

E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein ligase; EMCV, encephalomyocarditis virus; HAV, hepatitis A virus; PAGE, polyacrylamide gel electrophoresis. In recent years it has been discovered that E3␣ and Ubr1p can catalyze the ubiquitination of proteins lacking destabilizing N-terminal amino acids (type III substrates). Ribonuclease S, the subtilisn-derived fragment of ribonuclease A, has a stabilizing serine N terminus, but it is known to be a substrate for mammalian E3␣-dependent ubiquitin-protein conjugate synthesis [20] This indicates these E3 enzymes can recognize substrate proteins through associations with other types of structural elements. The identification of two substrate proteins recognized by E3␣, both of which contain precisely mapped, internal sequences known to be required for E3␣-dependent ubiquitin conjugation, provides excellent model systems for detailed studies of the interactions that take place between E3␣ and substrate proteins lacking a destabilizing N-terminal amino acid. Lawson, unpublished results. 4 The HsUbc2b is identical in sequence to its rabbit ortholog [27] and is functionally indistinguishable from the “a” isoform [26]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION