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USP32 impairs SLC35F2 protein stability. (A) Exogenous protein levels of SLC35F2 in HEK293 cells were analyzed upon transfection with increasing concentrations of HA-USP32 along with Myc-SLC35F2. Western blot was performed to determine exogenous SLC35F2 expression. (B) The effect of USP32 on endogenous SLC35F2 protein expression was determined in HeLa cells transfected with increasing concentrations of HA-USP32. Western blot analysis was performed to determine endogenous SLC35F2 protein. (C) HEK293 cells transfected with increasing concentrations of USP32 C743A, along with Myc-SLC35F2 to check the effect of the catalytic mutant of USP32 (USP32 C743A). Western blot analysis was performed with the indicated antibodies. (D) The effect of USP32 C743A on endogenous SLC35F2 protein was analyzed upon transfection with increasing concentrations of USP32 in HeLa cells. Western blot analysis was performed with SLC35F2-specific antibodies. (E) HEK293 cells were co-transfected with Myc-SLC35F2 alone or in combination with USP32 sgRNA1 or USP32 sgRNA2. Western blot analysis was used to determine the exogenous SLC35F2 protein level. The graph in the right panel represents expression of the Myc-SLC35F2 protein. (F) Endogenous SLC35F2 protein levels were measured in the presence of sgRNAs targeting USP32 by western blot analysis in HeLa cells. The graph in the right panel represents endogenous expression of SLC35F2 protein. (G) Validation of single cell-derived USP32 knockout clones in HEK293 cells by western blot analysis. Cell lysates were collected and immunoblotted with respective antibodies to analyze endogenous expression of USP32 and SLC35F2. (H) HEK293_USP32KO cells were transfected with either HA-USP32 or HA-USP32 C743A plasmids to measure the reconstitution effect of USP32 on endogenous SLC35F2 protein by western blot analysis. (I) HEK293 cells were co-transfected with Myc-SLC35F2 alone or in combination with HA-USP32 or in the presence of sgRNAs targeting USP32 to check the reconstitution effect of USP32 on exogenous SLC35F2 expression by western blot analysis. (J) Endogenous interaction between USP32 and SLC35F2 was analyzed in HeLa cells. Cell lysates from HeLa cells were immunoprecipitated with USP32-or SLC35F2-specific antibodies and immunoblotted with indicated antibodies. (K) HeLa cells were subjected to Duolink PLA assay and stained with mentioned antibodies. Scale bar = 25 µm.
Source publication
Background: The most commonly preferred chemotherapeutic agents to treat cancers are small-molecule drugs. However, the differential sensitivity of various cancer cells to small molecules and untargeted delivery narrow the range of potential therapeutic applications. The mechanisms responsible for drug resistance in a variety of cancer cells are al...
Contexts in source publication
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... determine whether USP32 destabilizes SLC35F2, we transfected HA-USP32 in a dosedependent manner. We found that SLC35F2 was reduced in a dose-dependent manner at either exogenous ( Figure 3A) or endogenous levels ( Figure 3B). In contrast, dose-dependent increase of catalytic mutant USP32 (HA-USP32 C743A) had no impact on SLC35F2 levels at either exogenous ( Figure 3C) or endogenous levels ( Figure 3D). ...
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... determine whether USP32 destabilizes SLC35F2, we transfected HA-USP32 in a dosedependent manner. We found that SLC35F2 was reduced in a dose-dependent manner at either exogenous ( Figure 3A) or endogenous levels ( Figure 3B). In contrast, dose-dependent increase of catalytic mutant USP32 (HA-USP32 C743A) had no impact on SLC35F2 levels at either exogenous ( Figure 3C) or endogenous levels ( Figure 3D). ...
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... found that SLC35F2 was reduced in a dose-dependent manner at either exogenous ( Figure 3A) or endogenous levels ( Figure 3B). In contrast, dose-dependent increase of catalytic mutant USP32 (HA-USP32 C743A) had no impact on SLC35F2 levels at either exogenous ( Figure 3C) or endogenous levels ( Figure 3D). A DUB-knockout library kit consisting of sgRNAs individually targeting an entire set of genes encoding USPs along with Cas9 were co-transfected using the Lipofectamine 2000 in HeLa cells. ...
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... found that SLC35F2 was reduced in a dose-dependent manner at either exogenous ( Figure 3A) or endogenous levels ( Figure 3B). In contrast, dose-dependent increase of catalytic mutant USP32 (HA-USP32 C743A) had no impact on SLC35F2 levels at either exogenous ( Figure 3C) or endogenous levels ( Figure 3D). A DUB-knockout library kit consisting of sgRNAs individually targeting an entire set of genes encoding USPs along with Cas9 were co-transfected using the Lipofectamine 2000 in HeLa cells. ...
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... transfected USP32 sgRNAs to validate the knockdown effect of USP32 on SLC35F2 protein. SLC35F2 was upregulated when USP32 was depleted by sgRNA2 at either exogenous ( Figure 3E, lane 4) or endogenous levels ( Figure 3F, lane 3). We previously generated several stable USP32 knockouts in HEK293 cells using sgRNA2 [22] and validated its knockout efficiency using western blot ( Figure 3G). ...
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... transfected USP32 sgRNAs to validate the knockdown effect of USP32 on SLC35F2 protein. SLC35F2 was upregulated when USP32 was depleted by sgRNA2 at either exogenous ( Figure 3E, lane 4) or endogenous levels ( Figure 3F, lane 3). We previously generated several stable USP32 knockouts in HEK293 cells using sgRNA2 [22] and validated its knockout efficiency using western blot ( Figure 3G). ...
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... was upregulated when USP32 was depleted by sgRNA2 at either exogenous ( Figure 3E, lane 4) or endogenous levels ( Figure 3F, lane 3). We previously generated several stable USP32 knockouts in HEK293 cells using sgRNA2 [22] and validated its knockout efficiency using western blot ( Figure 3G). SLC35F2 was upregulated in most of the USP32 knockouts clones ( Figure 3G). ...
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... previously generated several stable USP32 knockouts in HEK293 cells using sgRNA2 [22] and validated its knockout efficiency using western blot ( Figure 3G). SLC35F2 was upregulated in most of the USP32 knockouts clones ( Figure 3G). High SLC35F2 level was observed in clone #3 (hereafter HEK293_USP32KO) than mock ( Figure 3G, lane 4) and used for further experiments. ...
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... was upregulated in most of the USP32 knockouts clones ( Figure 3G). High SLC35F2 level was observed in clone #3 (hereafter HEK293_USP32KO) than mock ( Figure 3G, lane 4) and used for further experiments. The overexpression of wild-type USP32 ( Figure 3H, lane 3) but not catalytic mutant ( Figure 3H, lane 4) reverses the SLC35F2 protein stabilization in HEK293_USP32KO. ...
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... SLC35F2 level was observed in clone #3 (hereafter HEK293_USP32KO) than mock ( Figure 3G, lane 4) and used for further experiments. The overexpression of wild-type USP32 ( Figure 3H, lane 3) but not catalytic mutant ( Figure 3H, lane 4) reverses the SLC35F2 protein stabilization in HEK293_USP32KO. Similarly, we reconstituted USP32 in USP32-depleted cells and analyzed the Myc-SLC35F2 levels in HEK293 cells. ...
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... SLC35F2 level was observed in clone #3 (hereafter HEK293_USP32KO) than mock ( Figure 3G, lane 4) and used for further experiments. The overexpression of wild-type USP32 ( Figure 3H, lane 3) but not catalytic mutant ( Figure 3H, lane 4) reverses the SLC35F2 protein stabilization in HEK293_USP32KO. Similarly, we reconstituted USP32 in USP32-depleted cells and analyzed the Myc-SLC35F2 levels in HEK293 cells. ...
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... we reconstituted USP32 in USP32-depleted cells and analyzed the Myc-SLC35F2 levels in HEK293 cells. USP32 overexpression destabilized SLC35F2 protein than mock ( Figure 3I, lane 3). The sgRNAs targeting USP32 upregulated SLC35F2 protein ( Figure 3I, lanes 4 and 5) and reconstitution reversed SLC35F2 stabilization ( Figure 3I, lanes 6 and 7). ...
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... overexpression destabilized SLC35F2 protein than mock ( Figure 3I, lane 3). The sgRNAs targeting USP32 upregulated SLC35F2 protein ( Figure 3I, lanes 4 and 5) and reconstitution reversed SLC35F2 stabilization ( Figure 3I, lanes 6 and 7). We also examined whether USP32 interacts with SLC35F2 by immunoprecipitation with USP32 or SLC35F2 antibodies. ...
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... overexpression destabilized SLC35F2 protein than mock ( Figure 3I, lane 3). The sgRNAs targeting USP32 upregulated SLC35F2 protein ( Figure 3I, lanes 4 and 5) and reconstitution reversed SLC35F2 stabilization ( Figure 3I, lanes 6 and 7). We also examined whether USP32 interacts with SLC35F2 by immunoprecipitation with USP32 or SLC35F2 antibodies. ...
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... also examined whether USP32 interacts with SLC35F2 by immunoprecipitation with USP32 or SLC35F2 antibodies. The results showed that USP32 co-precipitated with SLC35F2 and vice versa ( Figure 3J). Additionally, we demonstrated that USP32 and SLC35F2 interact with each other by Duolink PLA assay. ...
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... we demonstrated that USP32 and SLC35F2 interact with each other by Duolink PLA assay. As shown in Figure 3K, the in situ USP32-SLC35F2 interaction (PLA dots) was observed when USP32 and SLC35F2 were immunostained together but not when they were stained with USP32 or SLC35F2 antibody alone. These data suggest that USP32 interacts with SLC35F2 and negatively regulates its protein stability. ...
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... examine the antagonistic nature of USP32 on SLC35F2 protein level, we performed an endogenous ubiquitination assay in the presence of wild-type or mutant USP32. Wild-type USP32 promoted SLC35F2 ubiquitination ( Figure 4E, lane 3) but not catalytic mutant USP32 ( Figure 4E, lane 4). Likewise, transient knockdown of USP32 diminished SLC35F2 ubiquitination ( Figure 4E, lane 5). ...
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... expression patterns of USP32 and SLC35F2 in human breast (n = 21), colon (n = 32), and lung cancers (n = 32) obtained from ISU Abxis tissue microarray were subjected to immunohistochemistry staining. USP32 was highly upregulated in breast cancer while SLC35F2 were significantly lower in these tissues ( Figure 5F and Figure S3A). However, SLC35F2 was highly upregulated in colon ( Figure 5G and Figure S3B, lower panel) and lung cancer tissues ( Figure 5H and Figure S3C, lower panel) and relative low levels of USP32 were observed in the respective tissues ( Figure 5G-H and Figure S3B-C, upper panels). ...
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... was highly upregulated in breast cancer while SLC35F2 were significantly lower in these tissues ( Figure 5F and Figure S3A). However, SLC35F2 was highly upregulated in colon ( Figure 5G and Figure S3B, lower panel) and lung cancer tissues ( Figure 5H and Figure S3C, lower panel) and relative low levels of USP32 were observed in the respective tissues ( Figure 5G-H and Figure S3B-C, upper panels). Altogether, USP32 and SLC35F2 are negatively correlated with each other, a relationship that may provide a predictive tool for YM155-mediated DNA damage. ...
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... was highly upregulated in breast cancer while SLC35F2 were significantly lower in these tissues ( Figure 5F and Figure S3A). However, SLC35F2 was highly upregulated in colon ( Figure 5G and Figure S3B, lower panel) and lung cancer tissues ( Figure 5H and Figure S3C, lower panel) and relative low levels of USP32 were observed in the respective tissues ( Figure 5G-H and Figure S3B-C, upper panels). Altogether, USP32 and SLC35F2 are negatively correlated with each other, a relationship that may provide a predictive tool for YM155-mediated DNA damage. ...
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... was highly upregulated in breast cancer while SLC35F2 were significantly lower in these tissues ( Figure 5F and Figure S3A). However, SLC35F2 was highly upregulated in colon ( Figure 5G and Figure S3B, lower panel) and lung cancer tissues ( Figure 5H and Figure S3C, lower panel) and relative low levels of USP32 were observed in the respective tissues ( Figure 5G-H and Figure S3B-C, upper panels). Altogether, USP32 and SLC35F2 are negatively correlated with each other, a relationship that may provide a predictive tool for YM155-mediated DNA damage. ...
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... USP32 knockout showing high SLC35F2 level were reconstituted with wild-type and mutant USP32 to measure the USP32 and SLC35F2 levels. Reconstitution of wild-type USP32 but not mutant USP32 destabilized SLC35F2 protein in USP32-knockout MCF7 ( Figure 6H, lanes 3 and 4) and BT474 cells ( Figure 6I, lanes 3 and 4). We also used the USP32-knockout cells to validate the USP32 dependence for YM155 uptake. ...
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... USP32 knockout showing high SLC35F2 level were reconstituted with wild-type and mutant USP32 to measure the USP32 and SLC35F2 levels. Reconstitution of wild-type USP32 but not mutant USP32 destabilized SLC35F2 protein in USP32-knockout MCF7 ( Figure 6H, lanes 3 and 4) and BT474 cells ( Figure 6I, lanes 3 and 4). We also used the USP32-knockout cells to validate the USP32 dependence for YM155 uptake. ...
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... in this study, it was necessary to demonstrate the interaction between USP32 and SLC35F2 and its functional consequence on protein turnover of SLC35F2. We demonstrated by immunoprecipitation and immunofluorescence assays that USP32 destabilizes SLC35F2 protein level by interacting endogenously with it ( Figure 3). The strong interaction between USP32 and SLC35F2 motivated us to analyze the effect of USP32 on protein ubiquitination and turnover of SLC35F2. ...
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... determine whether USP32 destabilizes SLC35F2, we transfected HA-USP32 in a dosedependent manner. We found that SLC35F2 was reduced in a dose-dependent manner at either exogenous ( Figure 3A) or endogenous levels ( Figure 3B). In contrast, dose-dependent increase of catalytic mutant USP32 (HA-USP32 C743A) had no impact on SLC35F2 levels at either exogenous ( Figure 3C) or endogenous levels ( Figure 3D). ...
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... determine whether USP32 destabilizes SLC35F2, we transfected HA-USP32 in a dosedependent manner. We found that SLC35F2 was reduced in a dose-dependent manner at either exogenous ( Figure 3A) or endogenous levels ( Figure 3B). In contrast, dose-dependent increase of catalytic mutant USP32 (HA-USP32 C743A) had no impact on SLC35F2 levels at either exogenous ( Figure 3C) or endogenous levels ( Figure 3D). ...
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... found that SLC35F2 was reduced in a dose-dependent manner at either exogenous ( Figure 3A) or endogenous levels ( Figure 3B). In contrast, dose-dependent increase of catalytic mutant USP32 (HA-USP32 C743A) had no impact on SLC35F2 levels at either exogenous ( Figure 3C) or endogenous levels ( Figure 3D). A DUB-knockout library kit consisting of sgRNAs individually targeting an entire set of genes encoding USPs along with Cas9 were co-transfected using the Lipofectamine 2000 in HeLa cells. ...
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... found that SLC35F2 was reduced in a dose-dependent manner at either exogenous ( Figure 3A) or endogenous levels ( Figure 3B). In contrast, dose-dependent increase of catalytic mutant USP32 (HA-USP32 C743A) had no impact on SLC35F2 levels at either exogenous ( Figure 3C) or endogenous levels ( Figure 3D). A DUB-knockout library kit consisting of sgRNAs individually targeting an entire set of genes encoding USPs along with Cas9 were co-transfected using the Lipofectamine 2000 in HeLa cells. ...
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... transfected USP32 sgRNAs to validate the knockdown effect of USP32 on SLC35F2 protein. SLC35F2 was upregulated when USP32 was depleted by sgRNA2 at either exogenous ( Figure 3E, lane 4) or endogenous levels ( Figure 3F, lane 3). We previously generated several stable USP32 knockouts in HEK293 cells using sgRNA2 [22] and validated its knockout efficiency using western blot ( Figure 3G). ...
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... transfected USP32 sgRNAs to validate the knockdown effect of USP32 on SLC35F2 protein. SLC35F2 was upregulated when USP32 was depleted by sgRNA2 at either exogenous ( Figure 3E, lane 4) or endogenous levels ( Figure 3F, lane 3). We previously generated several stable USP32 knockouts in HEK293 cells using sgRNA2 [22] and validated its knockout efficiency using western blot ( Figure 3G). ...
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... was upregulated when USP32 was depleted by sgRNA2 at either exogenous ( Figure 3E, lane 4) or endogenous levels ( Figure 3F, lane 3). We previously generated several stable USP32 knockouts in HEK293 cells using sgRNA2 [22] and validated its knockout efficiency using western blot ( Figure 3G). SLC35F2 was upregulated in most of the USP32 knockouts clones ( Figure 3G). ...
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... previously generated several stable USP32 knockouts in HEK293 cells using sgRNA2 [22] and validated its knockout efficiency using western blot ( Figure 3G). SLC35F2 was upregulated in most of the USP32 knockouts clones ( Figure 3G). High SLC35F2 level was observed in clone #3 (hereafter HEK293_USP32KO) than mock ( Figure 3G, lane 4) and used for further experiments. ...
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... was upregulated in most of the USP32 knockouts clones ( Figure 3G). High SLC35F2 level was observed in clone #3 (hereafter HEK293_USP32KO) than mock ( Figure 3G, lane 4) and used for further experiments. The overexpression of wild-type USP32 ( Figure 3H, lane 3) but not catalytic mutant ( Figure 3H, lane 4) reverses the SLC35F2 protein stabilization in HEK293_USP32KO. ...
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... SLC35F2 level was observed in clone #3 (hereafter HEK293_USP32KO) than mock ( Figure 3G, lane 4) and used for further experiments. The overexpression of wild-type USP32 ( Figure 3H, lane 3) but not catalytic mutant ( Figure 3H, lane 4) reverses the SLC35F2 protein stabilization in HEK293_USP32KO. Similarly, we reconstituted USP32 in USP32-depleted cells and analyzed the Myc-SLC35F2 levels in HEK293 cells. ...
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... SLC35F2 level was observed in clone #3 (hereafter HEK293_USP32KO) than mock ( Figure 3G, lane 4) and used for further experiments. The overexpression of wild-type USP32 ( Figure 3H, lane 3) but not catalytic mutant ( Figure 3H, lane 4) reverses the SLC35F2 protein stabilization in HEK293_USP32KO. Similarly, we reconstituted USP32 in USP32-depleted cells and analyzed the Myc-SLC35F2 levels in HEK293 cells. ...
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... we reconstituted USP32 in USP32-depleted cells and analyzed the Myc-SLC35F2 levels in HEK293 cells. USP32 overexpression destabilized SLC35F2 protein than mock ( Figure 3I, lane 3). The sgRNAs targeting USP32 upregulated SLC35F2 protein ( Figure 3I, lanes 4 and 5) and reconstitution reversed SLC35F2 stabilization ( Figure 3I, lanes 6 and 7). ...
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... overexpression destabilized SLC35F2 protein than mock ( Figure 3I, lane 3). The sgRNAs targeting USP32 upregulated SLC35F2 protein ( Figure 3I, lanes 4 and 5) and reconstitution reversed SLC35F2 stabilization ( Figure 3I, lanes 6 and 7). We also examined whether USP32 interacts with SLC35F2 by immunoprecipitation with USP32 or SLC35F2 antibodies. ...
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... overexpression destabilized SLC35F2 protein than mock ( Figure 3I, lane 3). The sgRNAs targeting USP32 upregulated SLC35F2 protein ( Figure 3I, lanes 4 and 5) and reconstitution reversed SLC35F2 stabilization ( Figure 3I, lanes 6 and 7). We also examined whether USP32 interacts with SLC35F2 by immunoprecipitation with USP32 or SLC35F2 antibodies. ...
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... also examined whether USP32 interacts with SLC35F2 by immunoprecipitation with USP32 or SLC35F2 antibodies. The results showed that USP32 co-precipitated with SLC35F2 and vice versa ( Figure 3J). Additionally, we demonstrated that USP32 and SLC35F2 interact with each other by Duolink PLA assay. ...
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... we demonstrated that USP32 and SLC35F2 interact with each other by Duolink PLA assay. As shown in Figure 3K, the in situ USP32-SLC35F2 interaction (PLA dots) was observed when USP32 and SLC35F2 were immunostained together but not when they were stained with USP32 or SLC35F2 antibody alone. These data suggest that USP32 interacts with SLC35F2 and negatively regulates its protein stability. ...
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... examine the antagonistic nature of USP32 on SLC35F2 protein level, we performed an endogenous ubiquitination assay in the presence of wild-type or mutant USP32. Wild-type USP32 promoted SLC35F2 ubiquitination ( Figure 4E, lane 3) but not catalytic mutant USP32 ( Figure 4E, lane 4). Likewise, transient knockdown of USP32 diminished SLC35F2 ubiquitination ( Figure 4E, lane 5). ...
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... expression patterns of USP32 and SLC35F2 in human breast (n = 21), colon (n = 32), and lung cancers (n = 32) obtained from ISU Abxis tissue microarray were subjected to immunohistochemistry staining. USP32 was highly upregulated in breast cancer while SLC35F2 were significantly lower in these tissues ( Figure 5F and Figure S3A). However, SLC35F2 was highly upregulated in colon ( Figure 5G and Figure S3B, lower panel) and lung cancer tissues ( Figure 5H and Figure S3C, lower panel) and relative low levels of USP32 were observed in the respective tissues ( Figure 5G-H and Figure S3B-C, upper panels). ...
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... was highly upregulated in breast cancer while SLC35F2 were significantly lower in these tissues ( Figure 5F and Figure S3A). However, SLC35F2 was highly upregulated in colon ( Figure 5G and Figure S3B, lower panel) and lung cancer tissues ( Figure 5H and Figure S3C, lower panel) and relative low levels of USP32 were observed in the respective tissues ( Figure 5G-H and Figure S3B-C, upper panels). Altogether, USP32 and SLC35F2 are negatively correlated with each other, a relationship that may provide a predictive tool for YM155-mediated DNA damage. ...
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... was highly upregulated in breast cancer while SLC35F2 were significantly lower in these tissues ( Figure 5F and Figure S3A). However, SLC35F2 was highly upregulated in colon ( Figure 5G and Figure S3B, lower panel) and lung cancer tissues ( Figure 5H and Figure S3C, lower panel) and relative low levels of USP32 were observed in the respective tissues ( Figure 5G-H and Figure S3B-C, upper panels). Altogether, USP32 and SLC35F2 are negatively correlated with each other, a relationship that may provide a predictive tool for YM155-mediated DNA damage. ...
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... was highly upregulated in breast cancer while SLC35F2 were significantly lower in these tissues ( Figure 5F and Figure S3A). However, SLC35F2 was highly upregulated in colon ( Figure 5G and Figure S3B, lower panel) and lung cancer tissues ( Figure 5H and Figure S3C, lower panel) and relative low levels of USP32 were observed in the respective tissues ( Figure 5G-H and Figure S3B-C, upper panels). Altogether, USP32 and SLC35F2 are negatively correlated with each other, a relationship that may provide a predictive tool for YM155-mediated DNA damage. ...
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... USP32 knockout showing high SLC35F2 level were reconstituted with wild-type and mutant USP32 to measure the USP32 and SLC35F2 levels. Reconstitution of wild-type USP32 but not mutant USP32 destabilized SLC35F2 protein in USP32-knockout MCF7 ( Figure 6H, lanes 3 and 4) and BT474 cells ( Figure 6I, lanes 3 and 4). We also used the USP32-knockout cells to validate the USP32 dependence for YM155 uptake. ...
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... USP32 knockout showing high SLC35F2 level were reconstituted with wild-type and mutant USP32 to measure the USP32 and SLC35F2 levels. Reconstitution of wild-type USP32 but not mutant USP32 destabilized SLC35F2 protein in USP32-knockout MCF7 ( Figure 6H, lanes 3 and 4) and BT474 cells ( Figure 6I, lanes 3 and 4). We also used the USP32-knockout cells to validate the USP32 dependence for YM155 uptake. ...
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... in this study, it was necessary to demonstrate the interaction between USP32 and SLC35F2 and its functional consequence on protein turnover of SLC35F2. We demonstrated by immunoprecipitation and immunofluorescence assays that USP32 destabilizes SLC35F2 protein level by interacting endogenously with it ( Figure 3). The strong interaction between USP32 and SLC35F2 motivated us to analyze the effect of USP32 on protein ubiquitination and turnover of SLC35F2. ...
Citations
... The family of SLC proteins regulate multiple metabolic and signaling pathways. Some studies explored the role of solute carrier family SLC35 in various cancers [22,23]. SLC3A2 is upregulated in several cancers, such as human osteosarcoma, gastric cancer and breast cancer, and promotes tumor growth through ferroptosis signaling pathway [24,25]. ...
The role of SLC35A2 in breast cancer remains poorly understood, with limited available information on its significance. This study aimed to investigate the expression of SLC35A2 and clinicopathological variables in breast cancer patients. Immunohistochemical analysis of SLC35A2 protein was conductedon 40 adjacent non-neoplastic tissues and 320 breast cancer tissues. The study also assesed the association between SLC35A2 expression and breast cancer clinicopathological features of breast cancer, as well as its impact on overall survival. In comparison to adjacent non-neoplastic tissues, a significantly higher expression of SLC35A2 was observed in breast cancer tissues ( P = 0.020), and this expression was found to be independently correlated with HER2 positivity ( P = 0.001). Survival analysis indicated that patients with low SLC35A2 expression had a more favorable prognosis in HER2-positive subtype breast cancer (P = 0.017). These results suggest that SLC35A2 is overexpressed in breast cancer tissues compared to adjacent non-neoplastic tissues and may serve as a potential prognostic marker for HER2-positive subtype breast cancer. Furthermore, breast cancer patients with the HER2 positive subtype who exhibited decreased levels of SLC35A2 expression demonstrated improved long-term prognostic outcomes.
... Therefore, SLC35F2 could be a suitable biomarker for evaluating treatment strategies and prognosis in malignant tumors. Chandrasekaran et al. [19] demonstrated that ubiquitin-specific protease 32 (USP32) confers resistance to YM155 in cancer cells by destabilizing the protein level of SLC35F2. This finding suggests a novel approach to treat tumors that are resistant to small-molecule drugs. ...
Objective
To explore the potential clinical and prognostic significance of Homo sapiens solute carrier family 35 member F2 (SLC35F2) in the context of lung adenocarcinoma (LUAD).
Methods
The expression pattern of SLC35F2 in LUAD tissues and normal tissues was analyzed in The Cancer Genome Atlas (TCGA) datasets and validated in 12 pairs of fresh clinical LUAD tissues and their corresponding adjacent normal tissues using quantitative real-time PCR (qRT-PCR) and western blotting. Immunohistochemistry (IHC) was used to assess the protein expression of SLC35F2 in 60 paraffin-embedded LUAD tissues, and its associations with clinicopathological parameters were further examined. The prognostic significance of SLC35F2 mRNA expression was also evaluated using the Kaplan-Meier method, and Cox regression models in LUAD patients from the TCGA database. The potential utility of SLC35F2 as an indicator of recurrence or metastasis was explored through the follow-up of selected clinical LUAD cases. Lastly, gene set enrichment analysis (GSEA) was conducted to investigate the underlying biological mechanisms and signaling pathways.
Results
Bioinformatics analysis utilizing the TCGA database indicated that SLC35F2 mRNA exhibited heightened expression in LUAD tissues when compared to normal tissues. These findings were further substantiated through the examination of 12 pairs of clinical LUAD tissues and their corresponding adjacent normal tissues, employing qRT-PCR and western blotting techniques. IHC results from a cohort of 60 LUAD patients demonstrated an up-regulation of SLC35F2 in 38 out of 60 individuals (63.3 %), which exhibited a significant correlation with tumor size, lymph node metastasis, and clinical stage (all P < 0.05). Both the Kaplan-Meier curve and the Cox proportional hazard analyses indicated a strong association between the up-regulation of SLC35F2 mRNA expression and unfavorable overall survival (OS) in patients with LUAD, as observed in the TCGA datasets (P < 0.05). The follow-up findings from select clinical LUAD cases provided evidence that the expression of SLC35F2 could serve as a dependable biomarker for monitoring the recurrence or metastasis. Additionally, the GSEA highlighted the enrichment of apoptosis, adhesion, small cell lung cancer (SCLC), and p53 signaling pathways in the subgroup of LUAD patients with elevated SLC35F2 expression.
Conclusion
SLC35F2 exhibited an up-regulated in both mRNA and protein expression, rendering it a valuable independent prognostic indicator for patients diagnosed with LUAD.
... Overcoming drug resistance and enhancing tumor sensitivity to drugs are crucial challenges in the treatment of HCC [43]. Recent studies have discovered that USP32 contributes to drug resistance in cancer: In several kinds of cancer cells, USP32 can confer cell resistance to a small molecule inhibitor YM155 (Sepantronium bromide) through promoting the degradation of SLC35F2, a solutecarrier protein that is essential for the uptake of YM155 [44]. In GISTs, USP32 protects Ras-related protein Rab-35 (Rab35) from proteasomal degradation, leading to tumor resistance to Imatinib, a tyrosine kinase inhibitor that is wildly used to combat GISTs [11]. ...
Background
Ubiquitin-specific protease 32 (USP32) is a highly conserved gene that promotes cancer progression. However, its role in hepatocellular carcinoma (HCC) is not well understood. The aim of this project is to explore the clinical significance and functions of USP32 in HCC.
Methods
The expression of USP32 in HCC was evaluated using data from TCGA, GEO, TISCH, tissue microarray, and human HCC samples from our hospital. Survival analysis, PPI analysis and GSEA analysis were performed to evaluate USP32-related clinical significance, key molecules and enrichment pathways. Using the ssGSEA algorithm and TIMER, we investigated the relationships between USP32 and immune infiltrates in the TME. Univariate and multivariate Cox regression analyses were then used to identify key USP32-related immunomodulators and constructed a USP32-related immune prognostic model. Finally, CCK8, transwell and colony formation assays of HCC cells were performed and an HCC nude mouse model was established to verify the oncogenic role of USP32.
Results
USP32 is overexpressed in HCC and its expression is an independent predictive factor for outcomes of HCC patients. USP32 is associated with pathways related to cell behaviors and cancer signaling, and its expression is significantly correlated with the infiltration of immune cells in the TME. We also successfully constructed a USP32-related immune prognostic model using 5 genes. Wet experiments confirmed that knockdown of USP32 could repress the proliferation, colony formation and migration of HCC cells in vitro and inhibit tumor growth in vivo.
Conclusion
USP32 is highly expressed in HCC and closely correlates with the TME of HCC. It is a potential target for improving the efficacy of chemotherapy and developing new strategies for targeted therapy and immunotherapy in HCC.
... We queried USP32 in human tissues through the Human Protein Atlas website, the testis showed the greatest RNA expression of USP32 (Fig. 3). USP32 is upregulated in a variety of cancers, including small cell lung cancer [24], gastric cancer [25,26], breast cancer [23,27,28], epithelial ovarian cancer [29], glioblastoma [30], gastrointestinal stromal tumor [31], pancreatic duct adenocarcinoma [32] and acute myeloid leukemia [33]. Endogenous USP32 is found in the cytoplasm and membrane, according to the findings of a subcellular separation experiment [34] and a fluorescence protection experiment [23]. ...
... This is consistent with earlier findings that USP32 is an active membrane-bound ubiquitin protease [24]. In the investigation of drug resistance in tumor cells, USP32, a membrane protein, can result in resistance to the anticancer medication YM155 by interfering with the steady expression of SLC35F2 [28]. Subcellular localization studies also show that USP32 may be co-located with Golgi [23], and some studies have found that USP32 can regulate the participation of small GTPase Rab7 in Golgi endosome selection [35]. ...
An essential protein regulatory system in cells is the ubiquitin-proteasome pathway. The substrate is modified by the ubiquitin ligase system (E1-E2-E3) in this pathway, which is a dynamic protein bidirectional modification regulation system. Deubiquitinating enzymes (DUBs) are tasked with specifically hydrolyzing ubiquitin molecules from ubiquitin-linked proteins or precursor proteins and inversely regulating protein degradation, which in turn affects protein function. The ubiquitin-specific peptidase 32 (USP32) protein level is associated with cell cycle progression, proliferation, migration, invasion, and other cellular biological processes. It is an important member of the ubiquitin-specific protease family. It is thought that USP32, a unique enzyme that controls the ubiquitin process, is closely linked to the onset and progression of many cancers, including small cell lung cancer, gastric cancer, breast cancer, epithelial ovarian cancer, glioblastoma, gastrointestinal stromal tumor, acute myeloid leukemia, and pancreatic adenocarcinoma. In this review, we focus on the multiple mechanisms of USP32 in various tumor types and show that USP32 controls the stability of many distinct proteins. Therefore, USP32 is a key and promising therapeutic target for tumor therapy, which could provide important new insights and avenues for antitumor drug development. The therapeutic importance of USP32 in cancer treatment remains to be further proven. In conclusion, there are many options for the future direction of USP32 research.
... The sequential activation of ubiquitination pathway by E1 (ubiquitin-activating enzyme), E2 (Ubiquitin conjugases) and E3 (ubiquitin ligases) plays an important role in promoting protein degradation by ubiquitination [9]. SLC35F2 protein turnover is regulated in cancer cells through proteasomal degradation [10]. Recently, we reported that USP32 promotes ER-associated SLC35F2 protein degradation in breast cancer cells and subsequently resulted in acquisition of YM155 drug resistance [10]. ...
... SLC35F2 protein turnover is regulated in cancer cells through proteasomal degradation [10]. Recently, we reported that USP32 promotes ER-associated SLC35F2 protein degradation in breast cancer cells and subsequently resulted in acquisition of YM155 drug resistance [10]. Thus, identifying the factors, such as E3 ligases, might help to shed light on the molecular mechanisms involved in regulation of SLC35F2 protein abundance in cancer cells. ...
... Particularly, SLC35F2, an important member of this family has been implicated in cancer progression [4]. SLC35F2 is highly expressed in lung cancer, prostate cancer, breast cancer and bladder cancer and suggests that high expression of SLC35F2 is associated with cancer progression [2,4,10,18,19]. We previously demonstrated that the regulation of SLC35F2 protein abundance is associated with cancer progression [10]. However, the molecular mechanism regulating SLC35F2 protein turnover is still not completely understood. ...
Background:
The solute carrier family 35 F2 (SLC35F2), belongs to membrane-bound carrier proteins that control various physiological functions and are activated in several cancers. However, the molecular mechanism regulating SLC35F2 protein turnover and its implication in cancer progression remains unexplored. Therefore, screening for E3 ligases that promote SLC35F2 protein degradation is essential during cancer progression.
Methods:
The immunoprecipitation and Duolink proximity ligation assays (PLA) were used to determine the interaction between APC/CCdh1 and SLC35F2 proteins. A CRISPR/Cas9-mediated knockdown and rescue experiment were used to validate the functional significance of APC/CCdh1 on SLC35F2 protein stabilization. The ubiquitination function of APC/CCdh1 on SLC35F2 protein was validated using in vitro ubiquitination assay and half-life analysis. The role of APC/CCdh1 regulating SLC35F2-mediated tumorigenesis was confirmed by in vitro oncogenic experiments in HeLa cells.
Results:
Based on the E3 ligase screen and in vitro biochemical experiments, we identified that APC/CCdh1 interacts with and reduces SLC35F2 protein level. APC/CCdh1 promotes SLC35F2 ubiquitination and decreases the half-life of SLC35F2 protein. On the other hand, the CRISPR/Cas9-mediated depletion of APC/CCdh1 increased SLC35F2 protein levels. The mRNA expression analysis revealed a negative correlation between APC/CCdh1 and SLC35F2 across a panel of cancer cell lines tested. Additionally, we demonstrated that depletion in APC/CCdh1 promotes SLC35F2-mediated cell proliferation, colony formation, migration, and invasion in HeLa cells.
Conclusion:
Our study highlights that APC/CCdh1 is a critical regulator of SLC35F2 protein turnover and depletion of APC/CCdh1 promotes SLC35F2-mediated tumorigenesis. Thus, we envision that APC/CCdh1-SLC35F2 axis might be a therapeutic target in cancer.
... Ubiquitin-specific protease 32 (USP32) is recognized as a new member of the ubiquitin-specific proteases subfamily. It alters protein stability and localization, thereby regulating their activity in different pathological stages of many human diseases, like cancer (6)(7)(8). So far, it has been reported that USP32 was over-expressed in lung and breast cancers, enhancing cellular proliferation and tissue metastasis (9). ...
Objective:
Gastric cancer is the fifth most common neoplasm and the fourth reason for mortality globally. Incidence rates are highly variable and dependent on risk factors, epidemiologic and carcinogenesis patterns. Previous studies reported that Helicobacter pylori (H. pylori) infection is one the strongest known risk factor for gastric cancer. USP32 is a deubiquitinating enzyme identified as a potential factor associated with tumor progression and a key player in cancer development. On the other hand, SHMT2 is involved in serine-glycine metabolism to support cancer cell proliferation. Both USP32 and SHMT2 are reported to be upregulated in many cancer types, including gastric cancer, but its complete mechanism is not fully explored yet. The present study explored possible mechanism of action of USP32 and SHMT2 in the progression of gastric cancer.
Materials and methods:
In this experimental study, Capsaicin (0.3 g/kg/day) and H. pylori infection combination was used to successfully initiate gastric cancer conditions in mice. It was followed by 40 and 70 days of treatment to establish initial and advanced conditions of gastric cancer.
Results:
Histopathology confirmed formation of signet ring cell and initiation of cellular proliferation in the initial gastric cancer. More proliferative cells were also observed. In addition, tissue hardening was confirmed in the advanced stage of gastric cancer. USP32 and SHMT2 showed progressive upregulated expression, as gastric cancer progress. Immunohistologically, it showed signals in abnormal cells and high-intensity signals in the advanced stage of cancer. In USP32 silenced tissue, expression of SHMT2 was completely blocked and reverted cancer development as evident with less abnormal cell in initial gastric cancer. Reduction of SHMT2 level to one-fourth was observed in the advanced gastric cancer stages of USP32 silenced tissue.
Conclusion:
USP32 had a direct role in regulating SHMT2 expression, which attracted therapeutic target for future treatment.
... Knockdown of USP32 significantly decreases the proliferation and migration rates of human small cell lung cancer [15]. Recently, a DUB-screening analysis performed on breast cancer cells revealed a resistance mechanism governed by USP32, which is related to YM155 (Sepantronium Bromide) resistance [16]. Nevertheless, the role of USP32 in AML remains obscure. ...
... USP32 is an ancient and highly conserved gene [25]. Genomic sequencing performed by Chandrasekaran et al. [16] revealed that the Tre2 (USP6) oncogene was derived from the chimeric fusion of two genes, USP32 and TBC1D3. Studies have demonstrated that USP32 acts as an oncogene, is expressed in many malignant tumors, and negatively impacts the survival outcome [14,15,26]. ...
Acute myeloid leukemia (AML) is a myeloid malignancy with generally high mortality. Although recent advances in AML research have revealed that circRNAs play significant roles in AML progression, our understanding of the leukemogenic mechanism of circRNAs remains very limited. In this study, increased expression of hsa_circ_0013880 was observed in bone marrow mononuclear cells (BMNCs) of AML patients. Overexpression of hsa_circ_0013880 promotes AML cell proliferation and migration and reduces cell apoptosis. Mechanistically, hsa_circ_0013880 could elevate the expression of USP32, a deubiquitinating enzyme that is highly expressed in the BMNCs of AML patients. Given the deubiquitination function of USP32, we further hypothesize that USP32 may mediate the malignant behaviors of AML cells by regulating the stability of Ras-related protein (Rap1b). At the molecular level, we find that silencing of USP32 increases ubiquitinated Rap1b. Overexpression of Rap1b restores the effects of USP32 knockdown, which further verifies our hypothesis. In addition, we propose another hypothesis that a potential regulatory network among hsa_circ_0013880, miR-148a-3p/miR-20a-5p and USP32 might exist in the development of AML, according to bioinformatics website predictions and our preliminary experimental verification. Overall, our findings will enrich the understanding of the hsa_circ_0013880/USP32/Rap1b axis in AML development, which may contribute to the development of novel therapeutic strategies for AML.
... In this study, we applied our recently developed CRISPR-based single-guide RNA (sgRNA) library targeting DUBs [21][22][23] to identify novel DUBs that might cause cisplatin-resistance in cancers. In parallel, we performed genome-wide screening for DUBs that regulate MAST1 protein abundance in cancer cells. ...
Background: Cisplatin is one of the frontline anticancer agents. However, development of cisplatin-resistance limits the therapeutic efficacy of cisplatin-based treatment. The expression of microtubule-associated serine/threonine kinase 1 (MAST1) is a primary factor driving cisplatin-resistance in cancers by rewiring the MEK pathway. However, the mechanisms responsible for MAST1 regulation in conferring drug resistance is unknown.
Methods: We implemented a CRISPR/Cas9-based, genome-wide, dual screening system to identify deubiquitinating enzymes (DUBs) that govern cisplatin resistance and regulate MAST1 protein level. We analyzed K48- and K63-linked polyubiquitination of MAST1 protein and mapped the interacting domain between USP1 and MAST1 by immunoprecipitation assay. The deubiquitinating effect of USP1 on MAST1 protein was validated using rescue experiments, in vitro deubiquitination assay, immunoprecipitation assays, and half-life analysis. Furthermore, USP1-knockout A549 lung cancer cells were generated to validate the deubiquitinating activity of USP1 on MAST1 abundance. The USP1-MAST1 correlation was evaluated using bioinformatics tool and in different human clinical tissues. The potential role of USP1 in regulating MAST1-mediated cisplatin resistance was confirmed using a series of in vitro and in vivo experiments. Finally, the clinical relevance of the USP1-MAST1 axis was validated by application of small-molecule inhibitors in a lung cancer xenograft model in NSG mice.
Results: The CRISPR/Cas9-based dual screening system identified USP1 as a novel deubiquitinase that interacts, stabilizes, and extends the half-life of MAST1 by preventing its K48-linked polyubiquitination. The expression analysis across human clinical tissues revealed a positive correlation between USP1 and MAST1. USP1 promotes MAST1-mediated MEK1 activation as an underlying mechanism that contributes to cisplatin-resistance in cancers. Loss of USP1 led to attenuation of MAST1-mediated cisplatin-resistance both in vitro and in vivo. The combined pharmacological inhibition of USP1 and MAST1 using small-molecule inhibitors further abrogated MAST1 level and synergistically enhanced cisplatin efficacy in a mouse xenograft model.
Conclusions: Overall, our study highlights the role of USP1 in the development of cisplatin resistance and uncovers the regulatory mechanism of MAST1-mediated cisplatin resistance in cancers. Co-treatment with USP1 and MAST1 inhibitors abrogated tumor growth and synergistically enhanced cisplatin efficacy, suggesting a novel alternative combinatorial therapeutic strategy that could further improve MAST1-based therapy in patients with cisplatin-resistant tumors.
... Genome-scale screening of the ubiquitin-specific protease subfamily for survivin protein using the DUB KO library Recently, we have reported the generation of a CRISPR-Cas9-mediated DUB KO library to screen putative DUBs for the protein of interest. 29,30 The single-guide RNAs (sgRNAs) targeting entire sets of genes encoding USP subfamilies along with Cas9 were co-transfected into HCT116 cells. A variation in endogenous survivin protein levels caused by the loss of function of a particular DUB was analyzed by western blotting ( Figure 1A). ...
Survivin is a component of the chromosomal passenger complex, which includes Aurora B, INCENP and Borealin, and is required for chromosome segregation and cytokinesis. We performed a genome-wide screen of deubiquitinating enzymes for survivin. For the first time, we report that USP19 has a dual role in the modulation of mitosis and tumorigenesis by regulating survivin expression. Our results found that USP19 stabilizes and interacts with survivin in HCT116 cells. USP19 deubiquitinates survivin protein and extends its half-life. We also found that USP19 functions as a mitotic regulator by controlling the downstream signaling of survivin protein. Targeted genome knockout verified that USP19 depletion leads to several mitotic defects, including cytokinesis failure. Additionally, USP19 depletion results in significant enrichment of apoptosis and reduces the growth of tumors in the mouse xenograft. We envision that simultaneous targeting of USP19 and survivin in oncologic drug development would increase therapeutic value and minimize redundancy.
... Hence, we screened the specific DUBs that can reverse protein degradation of PAH. To identify DUBs that regulate the expression of the PAH protein, we used our recently designed CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated-9)-based USP-knockout sgRNA library (Chandrasekaran et al. 2021;Das et al. 2020;Haq et al. 2022). This screen determined that USP19 regulates the protein expression of PAH. ...
... To elucidate the role of DUBs in regulating the stability of the PAH protein, we used our recently developed CRISPR/Cas9-based DUB knockout sgRNA library to screen for DUBs (Chandrasekaran et al. 2021;Das et al. 2020) for which loss of function confers a reduction or increase in the expression of the PAH protein. We ectopically expressed HA-tagged PAH and Cas9 along with single-guide RNAs (sgR-NAs) each targeting 50 DUB genes belonging to the USP family in HEK293 cells. ...
Phenylalanine hydroxylase (PAH) is the key enzyme in phenylalanine metabolism, deficiency of which is associated with the most common metabolic phenotype of phenylketonuria (PKU) and hyperphenylalaninemia (HPA). A bulk of PKU disease-associated missense mutations in the PAH gene have been studied, and the consequence of each PAH variant vary immensely. Prior research established that PKU-associated variants possess defects in protein folding with reduced cellular stability leading to rapid degradation. However, recent evidence revealed that PAH tetramers exist as a mixture of resting state and activated state whose transition depends upon the phenylalanine concentration and certain PAH variants that fail to modulate the structural equilibrium are associated with PKU disease. Collectively, these findings framed our understanding of the complex genotype–phenotype correlation in PKU. In the current study, we substantiate a link between PAH protein stability and its degradation by the ubiquitin-mediated proteasomal degradation system. Here, we provide an evidence that PAH protein undergoes ubiquitination and proteasomal degradation, which can be reversed by deubiquitinating enzymes (DUBs). We identified USP19 as a novel DUB that regulates PAH protein stability. We found that ectopic expression of USP19 increased PAH protein level, whereas depletion of USP19 promoted PAH protein degradation. Our study indicates that USP19 interacts with PAH and prevents polyubiquitination of PAH subsequently extending the half-life of PAH protein. Finally, the increase in the level of PAH protein by the deubiquitinating activity of USP19 resulted in enhanced metabolic function of PAH. In summary, our study identifies the role of USP19 in regulating PAH protein stability and promotes its metabolic activity.
Graphical abstract
Graphical highlights
1. E3 ligase Cdh1 promotes PAH protein degradation leading to insufficient cellular amount of PAH causing PKU.
2. A balance between E3 ligase and DUB is important to regulate the proteostasis of PAH.
3. USP19 deubiquitinates and stabilizes PAH further protecting it from rapid degradation.
4. USP19 increases the enzymatic activity of PAH, thus maintaining normal Phe levels.
