Inhibition of the ubiquitin-proteasome system in Alzheimer's disease - PubMed (original) (raw)

Inhibition of the ubiquitin-proteasome system in Alzheimer's disease

Y A Lam et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Alzheimer's disease is the most common cause of dementia in the elderly. Although several genetic defects have been identified in patients with a family history of this disease, the majority of cases involve individuals with no known genetic predisposition. A mutant form of ubiquitin, termed Ub(+1), has been selectively observed in the brains of Alzheimer's patients, including those with nonfamilial Alzheimer's disease, but it has been unclear why Ub(+1) expression should be deleterious. Here we show that Ub(+1) is an efficient substrate for polyubiquitination in vitro and in transfected human cells. The resulting polyubiquitin chains are refractory to disassembly by deubiquitinating enzymes and potently inhibit the degradation of a polyubiquitinated substrate by purified 26S proteasomes. Thus, expression of Ub(+1) in aging brain could result in dominant inhibition of the Ub-proteasome system, leading to neuropathologic consequences.

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Figures

Figure 1

Figure 1

Ub+1 is refractory to processing and activation. (A) Unique sequence of Ub+1. The sequences of the extreme C termini of Ub, Ub+1 (17), and Ub+1-H6 (see Materials and Methods) are shown. (B) Processing (Ub blots). (Left) Ub+1 was incubated (see Materials and Methods) with GST-UCH-D that had been preinactivated (or not) with _N-_ethyl maleimide (NEM) (as indicated). (Right) Control: the same enzyme processed Ub-CEP52, a chemically synthesized Ub-ribosomal fusion protein. (C) Activation. Increasing concentrations of Ub (○) or Ub+1 (●) were mixed with 2 μM 125I-Ub before initiating assays of E1-Ub thiol ester formation with purified E1 (see Materials and Methods).

Figure 2

Figure 2

Assembly of Ub+1-capped chains in mammalian cells (Ub blots). (A) Experiment. Human embryonic kidney 293T cells were transfected with plasmids specifying unmodified Ub+1 (lanes 2, 4, and 7) or Ub+1-H6 (lanes 3, 5, and 8). (Left) 200 μg of soluble extract protein was incubated with 10 μl of nickel beads; 10% of the starting lysate (lanes 2 and 3) and unbound fraction (lanes 4 and 5) were analyzed. The entire bound fraction deriving from 675 μg of lysate protein was analyzed in lanes 7 and 8. * denote cross-reacting bands that bound to the beads. Lanes 1 and 6, mixture of authentic polyUb chains. Ub+1-H6 (band 1) and presumptive Ub+1-H6-capped chains (bands 2–4) are indicated on the right. Conjs-conjugates. (B) Controls. Human 293T cells were transfected with empty vector (lane 2), vector specifying wild-type Ub (lane 3), and vector specifying Ub+1-H6 (lane 4). Soluble extracts were analyzed as in lanes 2 and 3 of A; lane 1, chain standard. The band in lanes 2 and 3, which comigrates with Ub+1-H6 and which does not bind to nickel beads (lanes 3 versus 5, A), is probably wild-type Ub2.

Figure 3

Figure 3

Ub+1-capped chains resist disassembly. (A) Synthesis of Ub5+1 (Coomassie-stained gel). The conjugation reaction (see Materials and Methods) was sampled at the indicated times. (B) Disassembly of Ub5+1 by isopeptidase T (+1 blot). First lane: Ub+1 standard. Next four lanes: Ub5+1 was incubated with increasing concentrations of purified isopeptidase T (left to right: none, 0.1 μM, 0.5 μM, 1 μM) as described in Materials and Methods. (C) Disassembly of Ub4 by isopeptidase T (Ub blot). Conditions were identical to last four lanes of B, except the chain was Ub4. (D) Disassembly of Ub4 versus Ub5+1 in mammalian cell lysate (Ub blots). Soluble lysate from 293T cells, prepared using DTT, was incubated with Ub4 or Ub5+1 for the indicated times (see Materials and Methods). First lane, polyUb chain standard. A trace of Ub4, present as a contaminant in Ub5+1, was rapidly disassembled in the Ub5+1 incubation.

Figure 4

Figure 4

Ub5+1 potently inhibits 26S proteasomes. Purified 26S proteasomes were incubated with 35S-Ub5 dihydrofolate reductase, with or without the indicated chain inhibitor (○, Ub4; ●, Ub5+1) as described in Materials and Methods. Initial rates of degradation are expressed relative to a control without inhibitor. The lines are fits assuming competitive inhibition (see Materials and Methods), with _K_i = 350 ± 30 nM (Ub5+1) or 1.2 ± 0.08 μM (Ub4). (Inset) Chain inhibitors (Coomassie-stained gel): 1 μg Ub4 (lane 1); 0.75 μg Ub5+1 (lane 2).

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