Biomedical subjects

A Clawson

Publications and source records attributed to A Clawson.

4 recordsLinked to original sources

PubMed1998

The proteasome activator 11 S regulator or PA28. Contribution By both alpha and beta subunits to proteasome activation.

The proteasome 11 S regulator (REG) consists of two homologous subunits, REGalpha and REGbeta. Each subunit is capable of activating the proteasome, and when combined, they form superactive REGalpha/REGbeta complexes. We have previously shown that a highly conserved loop in the REGalpha crystal structure is critical for proteasome activation. We now show that hetero-oligomers formed from REGalpha activation loop mutants and wild-type REGbeta or vice versa are partially active. By contrast, hetero-oligomers bearing mutations in the activation loops of REGalpha and REGbeta subunits are inactive, demonstrating that both alpha and beta subunits contribute to proteasome activation. We have also characterized REG proteins with mutations near or at their C termini. Partially active REGalpha(Y249C) and REGalpha(M247V) and an inactive REGalpha subunit bearing five additional C-terminal amino acids formed active hetero-oligomers with REGbeta. REGbeta subunits lacking 1, 2, or 9 C-terminal amino acids did not bind or activate the proteasome, but each of these mutants formed partially active hetero-oligomers with the monomer REGalpha(N50Y). However, hetero-oligomers formed from REG subunits lacking the last 14 amino acids were unable to bind the proteasome. Thus, C-terminal regions of both alpha and beta subunits are required for hetero-oligomers to bind the proteasome.

Amino Acid Sequence↗

PubMed1998

Proteasome activation by REG molecules lacking homolog-specific inserts.

The peptidase activities of eukaryotic proteasomes are markedly activated by the 11 S REG or PA28. The three identified REG subunits, designated alpha, beta, and gamma, differ significantly in sequence over a short span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant proteins REGalphaDeltai, REGbetaDeltai, and REGgammaDeltai. The purified recombinant proteins were then tested for their ability to oligomerize and activate the proteasome. Both REGalphaDeltai and REGgammaDeltai formed apparent heptamers and activated human red cell proteasomes to the same extent as their full-length counterparts. By contrast, REGbetaDeltai exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REGbeta protein. REGbetaDeltai was able to form hetero-oligomers with a single site, monomeric REGalpha mutant and with REGalphaDeltai. At low concentrations, the REGalphaDeltai/REGbetaDeltai hetero-oligomers stimulated the proteasome less than REGalpha/REGbeta oligomers formed from wild-type subunits, and the reduced activation by REGalphaDeltai/REGbetaDeltai was due to removal of the REGbeta insert, not the REGalpha insert. These studies demonstrate that the REGalpha and REGgamma inserts play virtually no role in oligomerization or in proteasome activation. By contrast, removal of REGbeta insert reduces binding of this subunit and REGalpha/REGbeta oligomers to proteasomes. On the whole, however, our findings show that REG inserts are not required for binding and activating the proteasome. We speculate that they serve to localize REG-proteasome complexes within cells, possibly by binding components in endoplasmic reticulum membranes.

Amino Acid Sequence↗

PubMed1998

Identification of an activation region in the proteasome activator REGalpha.

Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, alpha, beta, and gamma, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REGalpha. Most are monomers that produce functional proteasome activators when mixed with REGbeta subunits. Five REGalpha mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REGalpha heptamer shows that this region forms a loop at the base of each REGalpha subunit. One mutation in this loop (N146Y) yields a REGalpha heptamer that binds the proteasome as tightly as wild-type REGalpha but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REGbeta (N135Y) and REGgamma (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.

Amino Acid Sequence↗

PubMed1993

Relationship between forward head posture and diagnosed internal derangement of the temporomandibular joint.

The purpose of this study was to determine if a sample of patients with internal derangement of the temporomandibular joint had a significantly greater amount of forward head posture than did an age- and gender-matched control group. Twenty-two patients at a single head, neck, and temporomandibular joint treatment center, who had the diagnosis of internal derangement of the temporomandibular joint, were compared with a control group of volunteers. The angle of each patient's head position was measured from four photographs (two of the patient sitting and two of the patient standing) with a diagonal line drawn from the spinous process of the seventh cervical vertebra to the tragus of the ear and a horizontal line drawn perpendicular to a plumb line suspended from the ceiling. The angles measured from each of the four photographs were averaged, and this figure was paired with that generated from an age- and gender-matched person in the control group. Results indicated that there was not a significantly greater degree of forward head posture in the experimental group.

Adolescent↗