Biochemical Purification of Distinct Proteasome Subsets

Abstract

Proteasomes are intricate cellular proteases that are able to degrade many protein and peptide substrates in vitro. These particles are structurally complex; they are assembled from at least 14 small molecular mass polypeptide subunits to form mature 20S proteasomes. Recently, we demonstrated that proteasome subsets may be discriminated by serological criteria, and have found that subtle differences in the subunit composition of proteasomes can alter their cleavage specificity. Proteasome structural complexity is further enhanced when some proteasomes associate with additional proteins to form a 26S ATPand ubiquitin-dependent protease. Herein we confirm the existence of distinct cellular forms of proteasomes, and show that they differ in their hydrophobic characteristics. We have reproducibly purified, using solely biochemical techniques, distinct proteasome subsets similar to the serologically defined LMP2^+ and LMP2^- proteasomes. These proteasome subsets differ in their expression of at least three polypeptides, and both lack several additional polypeptides as compared to the serologically defined LMP2^+ and LMP2^- proteasomes. Finally, we demonstrate that these structurally unique proteasomes differ in their capacity to cleave a defined panel of fluorogenic peptide substrates. It appears that mammalian cells might recruit and modify proteasomes to perform distinct cellular tasks.

Proteasome gene upregulation: a possible mechanism for intestinal adaptation

“…There are a total of 14 different subunits that are duplicated and give rise to 7 subunits on each of the 4 rings [11][12][13]. The alpha rings are thought to provide structural support and aid the regulatory cap in restricting entrance to the beta rings, the site of proteolysis [14][15][16][17]. These enzymes digest the proteins into shorter peptides, which are then recycled by the cell.…”

Inhibition of Proteasome Activity Impairs Centrosome-dependent Microtubule Nucleation and Organization

“…Twenty microliters of collected fractions (250 l) were assayed according to standard protocols (Brown and Monaco, 1993). Twenty microliters of collected fractions (250 l) were assayed according to standard protocols (Brown and Monaco, 1993).…”

Molecular Machines for Protein Degradation

“…In mammals, γ‐interferon provokes the substitution of the three active β subunits (β1, β2 and β5) with three newly synthesised low‐molecular‐mass polypeptide (LMP) subunits termed β1i, β2i and β5i 51. 52 The incorporation of the γ‐interferon‐inducible subunits into the CP requires its de novo assembly and depends on the cell's development state and the tissue type 53. 54 Collectively, these γ‐interferon‐inducible subunits are referred to as the immunosubunits as they tune the CP for higher efficiency to generate specific antigenic peptides 55.…”