Figure 2. Identification of proteins targeted for degradation by
HCMV using an inhibitor-based proteomic screen. (A) Results of the
inhibitor-based screen. All 145 proteins downregulated >1.5
fold are plotted, with down-regulated proteins divided into 4 groups
using rescue ratios of >1.5 as cut-offs. The table shows
the number of proteins in each group. For rescue ratios, the denominator
(mock with drug)/mock was limited to a minimum of 1 to prevent
artificial ratio inflation. (B) Examples of positive controls from the
existing literature that were validated in this screen. (C) Examples of
degraded proteins rescued by both inhibitors (top panels), MG132 only
(middle panels), or bortezomib only (bottom panels).
Certain proteins exhibited a greater degree of rescue with MG132
compared to bortezomib (Figure 2B, yellow dots ). Of the 21
proteins only rescued >1.5 fold by MG132, 13 (62%)
exhibited bortezomib rescue ratios of >1.25 and
<1.5, suggesting that many of this group of proteins may
nevertheless be proteasomally degraded. These included the PDZ domain
containing protein 11 (PDZD11) and transcriptional repressor BEN Domain
Containing 3 (BEND3) (Figure S2, Tables S1-2). In contrast,
8/21 proteins appeared genuinely to be selectively rescued by MG132 but
not bortezomib (bortezomib rescue ratio <1.25), including the
fibroblast growth factor receptor Golgi Glycoprotein 1 (GLG1), E3 ligase
Neural Precursor Cell Expressed, Developmentally Down-Regulated 4
(NEDD4) and carbohydrate sulfotransferase 14 (CHST14) (Figure 2C
middle panel, Table S1 ). Similar data were obtained for treatments with
500 nM and 1 µM bortezomib, validating these findings (Figure
3 ). Interestingly, Gene Ontology annotation of all 8 proteins indicated
an association with either the cell membrane, the Golgi apparatus, or
vesicle secretion. Furthermore, comparison of data with our previous
study examining protein rescue by MG132 or leupeptin indicated that
GLG1, NEDD4 and CHST14 were also significantly rescued by treatment with
the lysosomal protease inhibitor leupeptin (Figure 3 ),
suggesting that a proportion of the proteins rescued by MG132 alone are
degraded lysosomally.
Of proteins exhibiting a greater degree of rescue with bortezomib
compared to MG132 (Figure 2A, purple dots ), 8/9 (89%)
exhibited MG132 rescue ratios >1.25 but <1.5
(examples in Figure S2, 2C bottom panel ), suggesting that the
majority of all proteins in this class were in fact rescued by both
inhibitors. The one exception was LIM domain-containing protein AJUBA,
whose MG132 rescue ratio was 1.16 in this data (Figure 2C bottom
panel ), but neared significance in our previous study (Table
S2 ); these differences may reflect relatively poor quantitation by only
two or one peptides respectively.