Shinpei Yamada

Atomic force microscopy studies of interaction of the 20S proteasome with supported lipid bilayers.

The 20S proteasome plays important roles in degradation of intracellular proteins. Mechanisms of its activation, its localization in cells, and its binding to biomembranes are not well understood. In this study, we used atomic force microscopy (AFM) to investigate interactions between the 20S proteasome and supported bilayers of various lipids in a buffer. We found that the 20S proteasome specifically bound to supported bilayers containing phosphatidylinositol (PI), but did not bind to supported bilayers containing phosphatidylcholine, phosphatidic acid or dioleoyltrimethylammonium propane. Binding of the 20S proteasomes had a high orientation; almost all were in a top view position. The specific and orientational binding of the 20S proteasome with PI may play important roles inside cells such as endoplasmic reticulum (ER) membrane. Use of AFM to study supported bilayers provides new information on ligand-receptor interactions.

Functional and biochemical characterization of the 20S proteasome in a yeast temperature-sensitive mutant, rpt6-1

BackgroundRpt6-1 is a thermosensitive yeast mutant with a deletion of a gene encoding a regulatory subunit of the 26S proteasome, RPT6, which is able to grow at 25°C but not at 37°C. In this study, peptidase activities, activation profiles, and the subunit composition of the 20S proteasome purified from the rpt6-1 mutant was characterized.ResultsThe 20S proteasome purified from rpt6-1 exhibited low levels of peptidase activities in the absence of activators, but nearly same activated activities in the presence of activators, suggesting a gating defect in the proteasome channel. Detailed analyses of the composition of the 20S proteasome through separation of all subunits by two-dimensional gel electrophoresis followed by identification of each subunit using MALDI-TOF-MS revealed that two subunits, α1 and α7, differed from those of wild-type cells in both electrophoretic mobility and pI values. The changes in these two α-subunits were apparent at the permissive temperature, but disappeared during stress response at the restrictive temperature. Interestingly, upon disappearance of these changes, the levels of peptidase activity of the 20S proteasome in the rpt6-1 mutant were restored as the wild-type. These results suggest that two different forms of the α-subunits, α1 and α7, block the proteasome channel in the rpt6-1 mutant.ConclusionTwo α-subunits (α1 and α7) of the 20S proteasome in the rpt6-1 mutant differed from their wild-type counterparts and peptidase activities were found to be lower in the mutant than in the wild-type strain.

Irreversible potent activation and reversible inhibition of trypsin-like activity of 20S proteasome purified from Xenopus oocytes by fatty acid.

Abstract The 20S proteasome purified from animal cells has various latent peptidase activities. Fatty acids such as linoleic, linolenic and oleic acids strongly activate both the chymotrypsin-type and peptidylglutamylpeptide (PGP) hydrolase-type activities, but have been reported to have little activation or inhibition of the trypsin-type activity. We show here that an increase of the fatty acid concentration produces activation of chymotrypsin-type and PGP hydrolase-type in a biphasic fashion: no effect until the threshold concentration and then a sharp activation. In contrast, the trypsin-type activity was markedly inhibited at low concentrations of fatty acid, slightly activated at higher concentrations, and inhibited again at even higher concentrations. The inhibition was removed when the concentration of fatty acid was reduced by dilution after pre-incubation with the fatty acid. As a result, the activation pattern became biphasic, which was identical to that of chymotrypsin-type and PGP hydrolase-type activities. These results suggest that in the chymotrypsin-type and PGP hydrolase-type peptidases fatty acids bind first to a class of sites without direct effect on the peptidase activity, but after saturation of this class it permits more fatty acid to bind to another class of sites involved in the activation. In the trypsin-type peptidase an additional class of fatty acid binding sites is uniquely present, which is involved in the enzyme inhibition. The dilution procedure described above removes the fatty acid molecules bound to the inhibition sites, but not the fatty acid molecules bound to the activation sites; this results in the fatty acid activation profile indistinguishable from that of the chymotrypsinand PGP hydrolase-type peptidases.

Activation of the 20S Proteasome of Xenopus Oocytes by SDS: Evidence for the Substrate-Induced Conformational Change Characteristic of Trypsin-Like Peptidase

Abstract The 20S proteasome of eukaryotic cells has at least three distinct peptidase activities (trypsin-like, chymotrypsin-like and peptidylglutamylpeptide (PGP) hydrolase activities). These peptidases are latent and require appropriate activators. SDS has been widely used as an activator of these peptidases, but the mechanism of its activation remains unresolved. In this study, we investigated the kinetics of the SDS-activated hydrolysis of the above three types of peptidase of the 20S proteasome purified from Xenopus oocytes. When the reaction was started by simultaneous adding both SDS and substrate, maximal rates of hydrolysis were reached after appreciable lag phases with the trypsin-type substrate [t-butyloxycarbonylLeu-Arg-Arg-4-methylcoumaryl-7-amide (Boc-LRR-MCA)], but no such lag phases were observed with the chymotrypsin-type and PGP hydrolase-type substrates [succinyl-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide (Suc-LLVY-MCA), and benzyloxycarbonyl-Leu-Leu-Glu-2-naphthylamide (Cbz-LLE-2NA), respectively]. Similarly, changes in the hydrolysis rate to a reduced level upon dilution of SDS occurred after an appreciable lag phase again in the trypsin-like peptidase, but not in the other types. The lag phase characteristic of the trypsin-like peptidase was dependent on the substrate concentration. Thus, the lag phase was less discernible at very low concentrations of the substrate (e.g. at concentrations in the order of 1/100 of the Km value), but became more conspicuous with the increases in the substrate concentration. This lag phase also vanished upon preincubation of the activator (SDS) for a short period of 5 sec. These results suggest that the formation of the enzyme-substrate complex in the trypsin-like reaction induces a conformational change in the enzyme which makes the SDS activator site(s) in an occluded form, reducing the rates of SDS binding and dissociation.

High affinity Zn2+ inhibitory site(s) for the trypsin-like peptidase of the 20S proteasome.

The effect of Zn(2+) on three major peptidase activities of the 20S proteasome purified from Xenopus oocytes was kinetically investigated. An extremely low concentration of Zn(2+) (microM range) strongly inhibited the trypsin-like activity of the 20S proteasome which was fully recoverable by the addition of EDTA. The concentration of Zn(2+) for half-maximum inhibition (K(0.5)) was 0.60 microM which was at least 10 times lower than that of any other divalent cation tested and essentially the same as for proteasomes purified from various other organisms indicating that the inhibition is highly Zn(2+)-specific, reversible, and common to the proteasome regardless of its source. Zn(2+) at concentrations below 100 microM instantaneously activated the chymotrypsin-like and PGPH activities, and the Zn(2+) concentration for half-maximum activation was found to be 42-48 microM. These activities were time-dependently inactivated by submillimolar concentrations of Zn(2+). The inactivation rates were dependent on the concentration of Zn(2+) and reached a maximum of 1.60-2.40 min(-1) for the three peptidase activities under the conditions used. The Zn(2+) concentration for half-maximum inactivation was found to be 0.70-1.23 mM. This time-dependent inactivation was not reversed by the addition of EDTA or DTT and might not be accompanied by the dissociation of subunits of the 20S proteasome indicating that all activities are inactivated by an identical phenomenon. These results reveal the three types of effects of Zn(2+) on the 20S proteasome.