Fig 2: 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.

Fig 3: 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, a-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.