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. 2010 Jan;6(1):e1000807.
doi: 10.1371/journal.pgen.1000807. Epub 2010 Jan 8.

Activation of mutant enzyme function in vivo by proteasome inhibitors and treatments that induce Hsp70

Laishram R Singh  1 , Sapna Gupta, Nicholaas H Honig, Jan P Kraus, Warren D Kruger
Affiliations

Activation of mutant enzyme function in vivo by proteasome inhibitors and treatments that induce Hsp70

Laishram R Singh et al. PLoS Genet. 2010 Jan.

Abstract

Missense mutant proteins, such as those produced in individuals with genetic diseases, are often misfolded and subject to processing by intracellular quality control systems. Previously, we have shown using a yeast system that enzymatic function could be restored to I278T cystathionine beta-synthase (CBS), a cause of homocystinuria, by treatments that affect the intracellular chaperone environment. Here, we extend these studies and show that it is possible to restore significant levels of enzyme activity to 17 of 18 (94%) disease causing missense mutations in human cystathionine beta-synthase (CBS) expressed in Saccharomyces cerevisiae by exposure to ethanol, proteasome inhibitors, or deletion of the Hsp26 small heat shock protein. All three of these treatments induce Hsp70, which is necessary but not sufficient for rescue. In addition to CBS, these same treatments can rescue disease-causing mutations in human p53 and the methylene tetrahydrofolate reductase gene. These findings do not appear restricted to S. cerevisiae, as proteasome inhibitors can restore significant CBS enzymatic activity to CBS alleles expressed in fibroblasts derived from homocystinuric patients and in a mouse model for homocystinuria that expresses human I278T CBS. These findings suggest that proteasome inhibitors and other Hsp70 inducing agents may be useful in the treatment of a variety of genetic diseases caused by missense mutations.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Functional rescue of mutant CBS by ethanol and hsp26Δ.
(A) A stationary phase culture of yeast (WY35) transformed with a plasmid expressing the indicated human CBS allele was diluted 1∶1000 in SC–Cys media with or without 4% ethanol and grown at 30°C for 24 h. Growth was determined by measuring OD at 600 nm. (B). Western analysis of two representative ethanol rescuable (T353M, T262M) and two non-rescuable (G307S, D376N) mutants. Cells were grown in SC+CYS media with or without ethanol and extracts were probed with indicated antibody. (C) CBS enzyme activity of each of the mutant alleles in the presence and absence of ethanol. (D) Growth of HSP26 (WY35) or hsp26Δ (LS2) yeast expressing the indicated CBS allele. (E) Western analysis of two representative hsp26Δ rescuable (T353M, A231L) and two non-rescuable (T191M, L101P) mutants. (F) CBS enzyme activity of each of the mutant alleles in the presence and absence of Hsp26.
Figure 2
Figure 2. Functional rescue of mutant human CBS expressed in S. cerevisiae by bortezomib treatment.
(A) A stationary phase culture of yeast (WY35) transformed with a plasmid expressing either WT (phCBS) or I278T (pI278T) human CBS was diluted 1∶1000 in SC–Cys media with the indicated amount of bortezomib and grown at 30°C for 24 h. Growth was determined by measuring OD at 600 nm. (B) WY35 pI278T and WY35 phCBS cells were grown in SC–Trp+Cys media either with or without 50 µM of bortezomib for 24 hours and extracts were prepared. Levels of CBS, α-tubulin, and Hsp70 and CBS activity were assessed as described in Materials and Methods. CBS units are nmole cystathionine formed/mg protein-hr. (C) Yeast strain WY35 phCBS, WY35 pI278T, LS3 phCBS, LS3 pI278T were grown at 30°C in the absence and presence of bortezomib and extracts were assessed for CBS activity and CBS protein. (D) Stationary phase cultures of yeast strain WY35 expressing the indicated allele of CBS were diluted 1∶1000 in SC–Cys media either with or without 50 µM of bortezomib and growth was monitored by OD600 after 24 h at 30°C. (E) Log phase cultures of WY35 grown in SC+Cys media with or without 50 µM of bortezomib expressing the indicated CBS allele were harvested, lystates prepared, and CBS enzyme activity was measured and expressed in units.
Figure 3
Figure 3. Functional rescue of mutant p53 and MTHFR in yeast.
(A) Yeast p53 tester strain yIG397 (HSP26), or LS2 (hsp26Δ) were transformed with an expression plasmid expressing the indicated p53 alleles. Stationary phase cultures of yeast expressing the wild type and indicated mutant were diluted 1∶1000 in SC-ura -ade media grown in 15 ml tubes with aeration at 30°C for 24 hours. Where indicated, 4% ethanol or 50 µM bortezomib were added to the media. (B) Yeast strain yIG397 carrying the indicated p53 alleles was grown in adenine containing media with or without 4% ethanol. Lysates were then probed by Western Blot with the indicated anti-bodies. (C) Yeast strains yIG397 and LS2 expressing the indicated p53 alleles were grown in adenine containing media supplemented with 4% ethanol when indicated. Lysates were prepared and immunoprecipitation was performed with anti-p53 antibody and Hsp26 present in the immune complexes was analyzed by immunoblot. (D) Yeast MTHFR tester strain XSY3-1A (met11Δ) were transformed with expression plasmids expressing the indicated MTHFR allele.
Figure 4
Figure 4. Functional rescue of mutant CBS by proteosome inhibitors in mammalian cells.
(A) Restoration of CBS enzyme activity in patient fibroblasts. Primary fibroblast cells expressing the indicated CBS allele were grown to 80% confluency at which time MG132 (18 µM) was added. After seven hours protein was harvested and Hsp70, CBS, and actin protein levels were measured by immunoblot. CBS activity was measured and activity is expressed as nmole cystathionine formed/mg protein-hr. Note that the G307S cell line is a transformed lymphoid cell line. (B) Effect of intravenous bortezomib treatment on Tg-I278T Cbs−/− mice. Tg-I278T Cbs−/− mice were kept on zinc water for 10 days to induce transgene expression and then either injected with bortezomib (30 µg) or saline by tail vein injection. After 17 hours mice were sacrificed and tissues harvested. Immunoblot analysis of CBS in liver and kidney extracts from bortezomib treated “B” and non-treated (NT) I278T mice. Actin was used as loading control. CBS activity was measured and activity is expressed as nmole cystathionine formed/mg protein-hr. Mice labeled “No Tg” are Cbs−/ − animals lacking human CBS. Lane labeled “C” contains extract from a Tg-I278T Cbs+/− animal. Bar graph shows Liver CBS activity in bortezomib treated (n = 4) and non-treated (n = 4) Tg-I278T Cbs−/−mice was compared with CBS activity in a non-treated mouse containing normal hCBS. Statistical significance was calculated by t test. Results are represented in terms of mean±SE. *P<0.05.
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