The 26S proteasome is a multisubunit protease responsible for regulated proteolysis in eukaryotic cells. It comprises one catalytic 20S proteasome and two axially positioned 19S regulatory complexes. The 20S proteasome is composed of 28 subunits arranged in a cylindrical particle as four heteroheptameric rings, alpha1-7beta1-7beta1-7alpha1-7 (refs 4, 5), but the mechanism responsible for the assembly of such a complex structure remains elusive. Here we report two chaperones, designated proteasome assembling chaperone-1 (PAC1) and PAC2, that are involved in the maturation of mammalian 20S proteasomes. PAC1 and PAC2 associate as heterodimers with proteasome precursors and are degraded after formation of the 20S proteasome is completed. Overexpression of PAC1 or PAC2 accelerates the formation of precursor proteasomes, whereas knockdown by short interfering RNA impairs it, resulting in poor maturation of 20S proteasomes. Furthermore, the PAC complex provides a scaffold for alpha-ring formation and keeps the alpha-rings competent for the subsequent formation of half-proteasomes. Thus, our results identify a mechanism for the correct assembly of 20S proteasomes.

Low-density lipoprotein receptor (LDLR) mRNA is unstable, but is stabilized upon extracellular signal-regulated kinase (ERK) activation, possibly through the binding of certain proteins to the LDLR mRNA 3′-untranslated region (UTR), although the detailed mechanism underlying this stability control is unclear. Here, using a proteomic approach, we show that proteins ZFP36L1 and ZFP36L2 specifically bind to the 3′-UTR of LDLR mRNA and recruit the CCR4-NOT-deadenylase complex, resulting in mRNA destabilization. We also show that the C-terminal regions of ZFP36L1 and ZFP36L2 are directly phosphorylated by p90 ribosomal S6 kinase, a kinase downstream of ERK, resulting in dissociation of the CCR4-NOT-deadenylase complex and stabilization of LDLR mRNA. We further demonstrate that targeted disruption of the interaction between LDLR mRNA and ZFP36L1 and ZFP36L2 using antisense oligonucleotides results in upregulation of LDLR mRNA and protein. These results indicate that ZFP36L1 and ZFP36L2 regulate LDLR protein levels downstream of ERK. Our results also show the usefulness of our method for identifying critical regulators of specific RNAs and the potency of antisense oligonucleotide-based therapeutics.

In globular proteins, there are intermolecular hydrogen bonds between protein and water molecules, and between water molecules, which are bound with the proteins, in addition to intramolecular hydrogen bonds. To estimate the contribution of these hydrogen bonds to the conformational stability of a protein, the thermodynamic parameters for denaturation and the crystal structures of five Thr to Val and five Thr to Ala mutant human lysozymes were determined. The denaturation Gibbs energy (DeltaG) of Thr to Val and Thr to Ala mutant proteins was changed from 4.0 to -5.6 kJ/mol and from 1.6 to -6.3 kJ/mol, respectively, compared with that of the wild-type protein. The contribution of hydrogen bonds to the stability (DeltaDeltaG(HB)) of the Thr and other mutant human lysozymes previously reported was extracted from the observed stability changes (DeltaDeltaG) with correction for changes in hydrophobicity and side chain conformational entropy between the wild-type and mutant structures. The estimation of the DeltaDeltaG(HB) values of all mutant proteins after removal of hydrogen bonds, including protein-water hydrogen bonds, indicates a favorable contribution of the intra- and intermolecular hydrogen bonds to the protein stability. The net contribution of an intramolecular hydrogen bond (DeltaG(HB[pp])), an intermolecular one between protein and ordered water molecules (DeltaG(HB[pw])), and an intermolecular one between ordered water molecules (DeltaG(HB[ww])) could be estimated to be 8. 5, 5.2, and 5.0 kJ/mol, respectively, for a 3 A long hydrogen bond. This result shows the different contributions to protein stability of intra- and intermolecular hydrogen bonds. The entropic cost due to the introduction of a water molecule (DeltaG(H)()2(O)) could be also estimated to be about 8 kJ/mol.

The structure of the tryptophan synthase ␣-subunit from Pyrococcus furiosus was determined by x-ray analysis at 2.0-Å resolution, and its stability was examined by differential scanning calorimetry. Although the structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium has been already determined, this is the first report of the structure of the ␣-subunit alone. The ␣-subunit from P. furiosus (Pf-␣-subunit) lacked 12 and 6 residues at the N and C termini, respectively, and one residue each in two loop regions as compared with that from S. typhimurium (St-␣-subunit), resulting in the absence of an N-terminal helix and the shortening of a C-terminal helix. The structure of the Pf-␣-subunit was essentially similar to that of the St-␣-subunit in the ␣ 2 ␤ 2 complex. The differences between both structures were discussed in connection with the higher stability of the Pf-␣-subunit and the complex formation of the ␣-and ␤-subunits. Calorimetric results indicated that the Pf-␣-subunit has extremely high thermostability and that its higher stability is caused by an entropic effect. On the basis of structural information of both proteins, we analyzed the contributions of each stabilization factor and could conclude that hydrophobic interactions in the protein interior do not contribute to the higher stability of the Pf-␣-subunit. Rather, the increase in ion pairs, decrease in cavity volume, and entropic effects due to shortening of the polypeptide chain play important roles in extremely high stability in Pf-␣-subunit.Prokaryotic tryptophan synthase, which catalyzes the last processes in the biosynthesis of tryptophan, is a multienzyme ␣ 2 ␤ 2 complex composed of nonidentical ␣-and ␤-subunits. The separate ␣-and ␤ 2 -subunits catalyze inherent reactions termed ␣ and ␤ reactions, respectively. When the ␣-and ␤ 2 -subunits combine to form the ␣ 2 ␤ 2 complex, the enzymatic activity of each subunit is stimulated by 1 to 2 orders of magnitude (1). The ␣ 2 ␤ 2 complex has been studied as an excellent model system for seeking answers to important questions in proteinprotein interaction, especially in multifunctional enzymes. In 1988 (2) the three-dimensional structure of the tryptophan synthase ␣ 2 ␤ 2 complex from Salmonella typhimurium was determined by x-ray analysis. However, the structure of the ␣-or ␤ 2 -subunit alone has not yet been determined. To elucidate the molecular basis of the mutual activation of the subunit interaction due to the formation of the ␣ 2 ␤ 2 complex, we need to know the structures of the ␣-or ␤ 2 -subunits alone as well as that of the complex. Although the crystallization of each subunit from S. typhimurium and Escherichia coli has been tried for many years (3), the report of the x-ray structure has not yet appeared. Recently, the structures of a number of proteins from hyperthermophiles have been successfully determined by x-ray analysis. This seems due to the facts that proteins from hyperthermophiles are unusually stable and more easily form better crystals. Therefore, the ...

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