Parallel alignments of designer yeast versus mammalian reporter assays.

A preliminary dose-dependent test for Fig 1C based on the readouts of ten indicated AR mutants (WT as control) in response to steroidal ligands. The OD600 values of liquid yeast cultures (without the presence of 3-AT) were measured 21 h post-incubation. Bars indicate mean ± s.d. (n = 2). (TIF)</p

Simulation of AR evolution by combining designer yeast with random mutagenesis.

(A) Characterization of the AR-LBD EP-PCR library. (B) Yeast clonal selection against compounds based on solid culture in 96-well plates without the presence of 3-AT. Ligands were 10−8 M DHT, 10−5 M PROG, 10−5 M E2, and 10−5 M CPA. The heatmap represents OD600 values of liquid cultures measured 21 h post-incubation. LBD-WT is indicated by pentacles; the × mark indicates blank wells. (C-D) Functional analysis of 19 identified AR mutations using both liquid (n = 6) and plate yeast assay in the presence of 3-AT against DHT 2 μM, E2 2 μM, PROG 2 μM, and CPA 2 μM. The incubation time was 60 h. Bars indicate mean ± s.d.</p

A dose-dependent assay to determine the appropriate concentrations of tested ligands supplemented in the yeast medium in the presence of 25 mM 3-AT.

Plate pictures were taken 48 h post-incubation. (TIF)</p

The functional analysis of 50 clinical AR mutants recorded in ARDB in response to steroidal ligands.

The “+” mark indicates a full response of the AR mutants to indicated ligands was observed in plate yeast assays compared with LBD-WT, while the “–” mark indicates the opposite. The “Partial” mark indicated a partial ligand-induced response of the AR mutants. The concentration of all ligands in the yeast assay were 2 μM. Plates were photographed 60 h after incubation. Thirteen mutants (R630Q, K631T, S648N, E666D, Q671R, L702H, V731M, S783N, Q799E, R847G, M887I, K911R and Q920R) behaved similarly to the wild type protein showing full response to the physiological ligand DHT and partial response to a high dose (2 μM) of E2. Eight mutants (Q641X, K721E, W742X, M750I, W752X, V758A, R787X, Q868X) were identified as loss-of function mutations failed to respond to any tested steroids. Mutations ending in X like Q641X indicated that a stop-codon was generated in this position resulting in the expression of a truncated AR protein.</p

Correlation of AR-mediated reporter activities between designer yeast and mammalian systems.

(A) Schematic representation of our designer yeast platform compared with the mammalian luciferase reporter system. (B) The list of 56 PCa-associated AR-LBD mutations accumulated in ARDB during a 22-year period (from 1990 to 2012). Top ten frequent mutations were marked in blue. (C), The Correlation analyses were based on fold changes in activation of AR-LBD mutants as normalized to the wild-type AR between the yeast and mammalian reporter systems, using the top ten frequent mutations in (B) against a variety of concentrations of DHT, E2, PROG, and CPA. Pearson’s correlation coefficient (R) and P-values were listed in the figures. Hormone concentrations in the yeast assay were DHT 10−8 M, E2 10−5 M, PROG 10−5 M, and CPA 10−5 M. Hormone concentrations in the human Hep3B reporter assay were DHT 10−9 M, E2 10−7 M, PROG 10−9 M, and CPA 10−7 M.</p

The plots of Fig 1C were created in R using the ‘ggpubr’ package.

The R codes as well as the relevant numerical values were described in this .docx file. (DOCX)</p

Sequential simulation of AR natural evolution.

(A) Two representative mutations per type are shown in yeast assays (n = 6) and luciferase assays (n = 3). OD600 values were measured 60 h post-incubation. The concentration of each ligand was 2 μM for yeast assays and 10 nM for luciferase assays. (B) The assessment of double-mutations identified in the second-round evolution, K778E/T878A and K778E/T878S, against tested steroidal ligands in liquid (n = 6) and plate yeast assays (with 25 mM 3-AT added). The concentration of each ligand was 2 μM. OD600 values were measured 48 h post-incubation. Plates were photographed 48 h post-incubation. Bars indicate mean ± s.d.</p

Summary of known and novel AR-LBD mutations based on protein structure.

Summary of known and novel AR-LBD mutations based on protein structure.</p

The distribution and abundance of secondary AR-LBD mutant library.

Distribution of secondary AR-LBD mutant library based on M750L, K778E, and E873D templates, respectively. Again, 94 high-ranking yeast colonies were selected for each ligand, followed by DNA isolation and sequencing to identify additional mutations. Ligands: 10−8 M DHT, 10−5 M PROG, 10−5 M E2, and 10−5 M CPA. The heatmap represents the OD600 values of liquid yeast cultures measured 21 h post-incubation (without the presence of 3-AT). (TIF)</p

Summary of sequenced AR mutations acquired in the secondary mutation library.

Summary of sequenced AR mutations acquired in the secondary mutation library.</p