W.W. de Jong

Structure and assembly of the 20S proteasome

The barrel-shaped 20S proteasome is one of the two components of a larger 26S particle, the multicatalytic 2000-kDa protease complex. The proteolytic sites are located in the inner chamber of the 20S particle and are only accessible via narrow entrances. This paper reviews the current knowledge concerning proteasome formation, proteolytic activities, structural aspects and assembly. Eukaryotic proteasomes are made up by four rings each of which contains seven different subunits occurring at fixed positions. While the outer rings contain α-type subunits, the inner ones comprise β-type subunits. The current assembly model for eukaryotic 20S proteasomes is based upon the detection of 13S and 16S intermediates, respectively, in addition to previous findings with archaebacterial and eubacterial proteasome assembly. The available data suggest a cooperative assembly of the α-type and β-type subunits into half proteasome-like complexes followed by dimerization into proteasomes. During or after dimerization of half proteasomes, the β-type subunits are processed. The prosequence of the β-type subunits is essential for the assembly process and prevents protease activity of immature proteasomes.

α-Crystallins, versatile stress-proteins

a-Crystallins owe their name to the fact that they are major eye lens proteins in vertebrates. They are abundantly present in the lens and have a prominent role in maintaining the transparency and refractile properties of the lens (reviewed in [1-3]). There are two types of related subunits, aA and c~B, each of about 20 kDa, of which aB is also expressed at significant levels in many different tissues outside the lens [4]. a-Crystallins exist as large homoor heteromeric complexes, containing about 30-40 subunits. Their tertiary and quaternary structures are unknown. Initially it was observed that, on the basis of sequence homology, a-crystallins belong to the family of small heat shock proteins (hsps) [5]. This family is characterized by the presence of an approximately 100 amino acid long conserved domain, often called the a-crystallin domain, a-Crystallins and small hsps are not only evolutionarily related but they also behave very similarly in many respects. They actually can form mixed complexes in tissues were both proteins are expressed [6, 7]. a-Crystallins, like other small hsps, have chaperone-like properties in that they can prevent stress-induced aggregation of proteins [8]. Furthermore, mammalian aB-crystallin is stress-inducible, and the presence of a-crystallins in cultured cells leads to an enhanced survival of these cells after a period of stress [9]. aB-Crystallin is also implicated in the pathogenesis of various degenerative diseases. In this mini review we will mainly discuss file structural and functional aspects of a-crystallins. For gene regulation [10], evolutionary relationships [11] and post-translational modifications [12] of a-crystallins we refer to other recent reviews.

The effect of αB-crystallin and Hsp27 on the availability of translation initiation factors in heat-shocked cells

Abstract.The mechanism of the translational thermotolerance provided by the small heat shock proteins (sHsps) αB-crystallin or Hsp27 is unknown. We show here that Hsp27, but not αB-crystallin, increased the pool of mobile stress granule-associated enhanced green fluorescent protein (EGFP)-eukaryotic translation initiation factor (eIF)4E in heat-shocked cells, as determined by fluorescence recovery after photobleaching. Hsp27 also partially prevented the sharp decrease in the pool of mobile cytoplasmic EGFP-eIF4G. sHsps did not prevent the phosphorylation of eIF2α by a heat shock, but promoted dephosphorylation during recovery. Expression of the C-terminal fragment of GADD34, which causes constitutive dephosphorylation of eIF2α, fully compensated for the stimulatory effect of αB-crystallin on protein synthesis in heat-shocked cells, but only partially for that of Hsp27. Our data show that sHsps do not prevent the inhibition of protein synthesis upon heat shock, but restore translation more rapidly by promoting the dephosphorylation of eIF2α and, in the case of Hsp27, the availability of eIF4E and eIF4G.

The molecular weight of the basic polypetide chain αB2 of α-crystallin

The molecular weights calculated from the amino acid sequences of the αA and αB chains of the lens protein α-crystallin differ only slightly (19830 and 20070, respectively). SDS gel electrophoresis of these chains and comparison with marker proteins yield apparent molecular weights of 19500 for αA and 22500 for αB. The discrepancy between the value of 22500 and the real molecular weight of 20070 for αB vanishes by the combined use of SDS and 6 M urea in the polyacrylamide gels.

Characterization of anti-crystallin autoantibodies in patients with cataract

Anti-crystallin autoantibodies have often been demonstrated in the serum of healthy persons and, especially, patients with cataract. In no case, however, have the specific crystallin subunits been identified against which such antibodies are directed. This information would be of particular interest in view of the recent finding that several crystallin subunits occur constitutively outside the lens. To fill this gap, we analysed the sera of 15 patients with mature cataract by means of 1- and 2-dimensional immunoblotting. The circulating antibodies turned out to be directed against several β- and γ-crystallin subunits. The types of subunits and the intensities of the responses varied considerably between patients. No or only occasional and very weak reactions were observed against the αA-, αB- and βB2-crystallin subunits. These are in fact the only crystallins at present known to occur outside the lens in mammals. Our findings thus indicate that anti-crystallin autoantibodies are specifically directed against those crystallins that appear to be lens-restricted, while immunological tolerance would exist for the extra-lenticularly occurring crystallins.

The primary structure of bovine lens leucine aminopeptidase: Complete amino acid sequence of the N-terminal cyanogen bromide fragment and site of limited tryptic digestion

Abstract The amino acid sequence of the N-terminal cyanogen bromide fragment of bovine lens leucine aminopeptidase has been determined. This fragment contains a total of 171 amino acid residues and has a calculated molecular weight of 18,637. The sequence data presented here represent the first report of primary structure determination of a member of the class of aminopeptidases. The single cleavage site produced by limited tryptic digestion of native leucine aminopeptidase was determined to be between arginine-137 and lysine-138 of the total amino acid sequence. The possible existence of distinct structural domains in leucine aminopeptidase is discussed.

Sequence of the first 68 residues of the αB2 chain of bovine α-crystallin

Summary The sequence of the N-terminal cyanogen bromide fragment of the αB2 chain of bovine α-crystallin was determined. Valuable results were obtained by the use of an extracellular protease from Staphylococcus aureus, cleaving specifically C-terminal of glutamyl residues, and the application of the solid-phase Edman degradation. A 55% sequence homology was observed with the N-terminal region of the αA2 chain of bovine α-crystallin.