Steven C. Johnston

Structural basis for the specificity of ubiquitin C-terminal hydrolases.

The release of ubiquitin from attachment to other proteins and adducts is critical for ubiquitin biosynthesis, proteasomal degradation and other cellular processes. De‐ubiquitination is accomplished in part by members of the UCH (ubiquitin C‐terminal hydrolase) family of enzymes. We have determined the 2.25 Å resolution crystal structure of the yeast UCH, Yuh1, in a complex with the inhibitor ubiquitin aldehyde (Ubal). The structure mimics the tetrahedral intermediate in the reaction pathway and explains the very high enzyme specificity. Comparison with a related, unliganded UCH structure indicates that ubiquitin binding is coupled to rearrangements which block the active‐site cleft in the absence of authentic substrate. Remarkably, a 21‐residue loop that becomes ordered upon binding Ubal lies directly over the active site. Efficiently processed substrates apparently pass through this loop, and constraints on the loop conformation probably function to control UCH specificity.

Crystal structure of a deubiquitinating enzyme (human UCH-L3) at 1.8 Å resolution

Ubiquitin C‐terminal hydrolases catalyze the removal of adducts from the C‐terminus of ubiquitin. We have determined the crystal structure of the recombinant human Ubiquitin C‐terminal Hydrolase (UCH‐L3) by X‐ray crystallography at 1.8 å resolution. The structure is comprised of a central antiparallel β‐sheet flanked on both sides by α‐helices. The β‐sheet and one of the helices resemble the well‐known papain‐like cysteine proteases, with the greatest similarity to cathepsin B. This similarity includes the UCH‐L3 active site catalytic triad of Cys95, His169 and Asp184, and the oxyanion hole residue Gln89. Papain and UCH‐L3 differ, however, in strand and helix connectivity, which in the UCH‐L3 structure includes a disordered 20 residue loop (residues 147‐166) that is positioned over the active site and may function in the definition of substrate specificity. Based upon analogy with inhibitor complexes of the papain‐like enzymes, we propose a model describing the binding of ubiquitin to UCH‐L3. The UCH‐L3 active site cleft appears to be masked in the unliganded structure by two different segments of the enzyme (residues 9‐12 and 90‐94), thus implying a conformational change upon substrate binding and suggesting a mechanism to limit non‐specific hydrolysis.

Structure of the proteasome activator REGα (PA28α)

The specificity of the 20S proteasome, which degrades many intracellular proteins, is regulated by protein complexes that bind to one or both ends of the cylindrical proteasome structure. One of these regulatory complexes, the 11S regulator (known as REG or PA28), stimulates proteasome peptidase activity, and enhances the production of antigenic peptides for presentation by class I molecules of the major histocompatibility complex (MHC),. The three REG subunits that have been identified, REGα, REGβ and REGγ (also known as the Ki antigen), share extensive sequence similarity, apart from a highly variable internal segment of 17–34 residues which may confer subunit-specific properties. REGα and REGβ preferentially form a heteromeric complex, although purified REGα forms a heptamer in solution and has biochemical properties similar to the heteromeric REGα/REGβ complex,. We have now determined the crystal structure of human recombinant REGα at 2.8 Å resolution. The heptameric barrel-shaped assembly contains a central channel that has an opening of 20 Å diameter at one end and another of 30 Å diameter at the presumed proteasome-binding surface. The binding of REG probably causes conformational changes that open a pore in the proteasome α-subunits through which substrates and products can pass.

Characterization of recombinant REGα, REGβ, and REGγ proteasome activators

Full-length cDNAs for three human proteasome activator subunits, called REGα, REGβ, and REGγ, have been expressed in Escherichia coli, and the purified recombinant proteins have been characterized. Recombinant α or γ subunits form heptameric species; recombinant β subunits are found largely as monomers or small multimers. Each recombinant REG stimulates cleavage of fluorogenic peptides by human red cell proteasomes. The pattern of activated peptide hydrolysis is virtually identical for REGα and REGβ. These two subunits, alone or in combination, stimulate cleavage after basic, acidic, and most hydrophobic residues in many peptides. Recombinant α and β subunits bind each other with high affinity, and the REGα/β heteromeric complex activates hydrolysis of LLVY-methylcoumaryl-7-amide (LLVY-MCA) and LLE-β-nitroanilide (LLE-βNA) more than REGα or REGβ alone. Using filter binding and gel filtration assays, recombinant REGγ subunits were shown to bind themselves but not α or β subunits. REGγ differs from REGα and REGβ in that it markedly stimulates hydrolysis of peptides with basic residues in the P1 position but only modestly activates cleavage of LLVY-MCA or LLE-βNA by the proteasome. REGγ binds the proteasome with higher affinity than REGα or REGβ yet with lower affinity than complexes containing both REGα and REGβ. In summary, each of the three REG homologs is a proteasome activator with unique biochemical properties.

Lincoln’s Decisionism and the Politics of Elimination:

Abraham Lincoln’s hallowed place in American memory is secure: He saved the Union, put an end to slavery, and was assassinated for these very successes. At the same time, Lincoln’s many undeniable achievements came at terrible—and lasting—democratic cost. Informed by the work of Carl Schmitt and Giorgio Agamben, this essay aspires to illuminate that cost by analyzing two cases where Lincoln exercised a sovereign decisionism—one involving the exile of Ohio politician Clement Vallandigham for publicly opposing the Civil War and the draft, a second involving the mass execution of Dakota Sioux Indians for daring to rise up and enact their own sovereign prerogatives during the war. This decisionism reveals Lincoln’s problematic resort to anti-political practices to deal with adversaries. Given the damage Lincoln did to American democracy, the essay also investigates what he might have done to make amends for it. Finally, it explores how Lincoln’s place in American history might be remembered more agonistically, architecturally speaking, on the Mall in Washington, D.C.