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Distinct OGT-Binding Sites Promote HCF-1 Cleavage.

Bhuiyan T, Waridel P, Kapuria V, Zoete V, Herr W - PLoS ONE (2015)

Bottom Line: HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle.Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage.These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrative Genomics, University of Lausanne, Génopode, Lausanne, Switzerland.

ABSTRACT
Human HCF-1 (also referred to as HCFC-1) is a transcriptional co-regulator that undergoes a complex maturation process involving extensive O-GlcNAcylation and site-specific proteolysis. HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by O-GlcNAc transferase (OGT), which thus displays an unusual dual enzymatic activity. OGT cleaves HCF-1 at six highly conserved 26 amino acid repeat sequences called HCF-1PRO repeats. Here we characterize the substrate requirements for OGT cleavage of HCF-1. We show that the HCF-1PRO-repeat cleavage signal possesses particular OGT-binding properties. The glutamate residue at the cleavage site that is intimately involved in the cleavage reaction specifically inhibits association with OGT and its bound cofactor UDP-GlcNAc. Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage. These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity.

No MeSH data available.


Related in: MedlinePlus

Region II CEE O-GlcNAcylation and HCF-1PRO-repeat proteolysis are independent OGT activities.(A) (Left) The full-length (FL) HCF-1rep1 precursor (band a) and the N-terminal cleavage product (band b) were purified from HEK 293 lysates via α-HA-epitope immunoprecipitation and visualized by Coomassie staining. The bands were analyzed for O-GlcNAcylation and phosphorylation sites by LC-MS/MS. (Right) Schematic representation of identified HCF-1rep1 O-GlcNAcylation (squares) and phosphorylation (yellow circles) sites in the uncleaved HCF-1rep1 precursor. The HCF-1 sequences covered by the analysis (residues 867–1071) and the engineered trypsin cleavage sites A933K and M951K are indicated below the diagram. Red and blue squares indicate confident (Mascot score > 23 & probability of localization > 70%) and potential (Mascot score 14–22 or probability of localization 50–70%) O-GlcNAcylation sites, respectively. Squares surrounded in black indicate previously identified sites [9]. The HCF-1 Region II CEE amino acid sequence spanning a peptide sequence used in subsequent analyses (underlined: 901–933K) is shown below the diagram.(B) Analysis of a representative Region II CEE peptide (901–933K sequence shown in A) by LC-MS/MS for proportions of different O-GlcNAcylated forms. The proportions of 901–933K peptides containing 0, 1, 2 or 3 attached O-GlcNAc moieties are given for each sample in percent. HCF-1rep1 constructs were synthesized in HEK 293 cells and peptides were derived from constructs containing wild-type (WT) or mutated (E10A, E10D, E10Q, E10S, T17–22A) HCF-1PRO repeats, or containing a deletion of the HCF-1PRO-repeat sequence (∆PRO). The results with WT precursor, E10A, and E10S were confirmed in a second independent experiment.(C) HCF-1rep1 O-GlcNAcylation is not fundamental for HCF-1PRO-repeat cleavage. In vitro cleavage activities of wild-type OGT (WT) and an O-GlcNAcylation compromised OGT mutant (D554H_H558D) on selected HCF-1rep1 substrates. Cleavage and O-GlcNAcylation activities of constructs containing the full-length HCF-1rep1 sequence (FL), or the Region II CEE (+II) or Region III (+III) sequences were analyzed by immunoblot using the indicated antibodies. We note that the lack of the OGT D554H_H558D O-GlcNAcylation activity results in differential mobility of the HCF-1rep1 cleavage products during electrophoresis. Prominent (●) and faint (⭕) cleavage products are indicated.
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pone.0136636.g004: Region II CEE O-GlcNAcylation and HCF-1PRO-repeat proteolysis are independent OGT activities.(A) (Left) The full-length (FL) HCF-1rep1 precursor (band a) and the N-terminal cleavage product (band b) were purified from HEK 293 lysates via α-HA-epitope immunoprecipitation and visualized by Coomassie staining. The bands were analyzed for O-GlcNAcylation and phosphorylation sites by LC-MS/MS. (Right) Schematic representation of identified HCF-1rep1 O-GlcNAcylation (squares) and phosphorylation (yellow circles) sites in the uncleaved HCF-1rep1 precursor. The HCF-1 sequences covered by the analysis (residues 867–1071) and the engineered trypsin cleavage sites A933K and M951K are indicated below the diagram. Red and blue squares indicate confident (Mascot score > 23 & probability of localization > 70%) and potential (Mascot score 14–22 or probability of localization 50–70%) O-GlcNAcylation sites, respectively. Squares surrounded in black indicate previously identified sites [9]. The HCF-1 Region II CEE amino acid sequence spanning a peptide sequence used in subsequent analyses (underlined: 901–933K) is shown below the diagram.(B) Analysis of a representative Region II CEE peptide (901–933K sequence shown in A) by LC-MS/MS for proportions of different O-GlcNAcylated forms. The proportions of 901–933K peptides containing 0, 1, 2 or 3 attached O-GlcNAc moieties are given for each sample in percent. HCF-1rep1 constructs were synthesized in HEK 293 cells and peptides were derived from constructs containing wild-type (WT) or mutated (E10A, E10D, E10Q, E10S, T17–22A) HCF-1PRO repeats, or containing a deletion of the HCF-1PRO-repeat sequence (∆PRO). The results with WT precursor, E10A, and E10S were confirmed in a second independent experiment.(C) HCF-1rep1 O-GlcNAcylation is not fundamental for HCF-1PRO-repeat cleavage. In vitro cleavage activities of wild-type OGT (WT) and an O-GlcNAcylation compromised OGT mutant (D554H_H558D) on selected HCF-1rep1 substrates. Cleavage and O-GlcNAcylation activities of constructs containing the full-length HCF-1rep1 sequence (FL), or the Region II CEE (+II) or Region III (+III) sequences were analyzed by immunoblot using the indicated antibodies. We note that the lack of the OGT D554H_H558D O-GlcNAcylation activity results in differential mobility of the HCF-1rep1 cleavage products during electrophoresis. Prominent (●) and faint (⭕) cleavage products are indicated.

Mentions: We purified full-length HCF-1rep1 with the engineered trypsin cleavage sites after transient synthesis in HEK 293 cells and analyzed the uncleaved precursor band (Fig 4A, band a), as well as the N-terminal cleavage product (band b) for O-GlcNAcylation. The identified O-GlcNAcylation sites of the HCF-1rep1 precursor and the N-terminal cleavage product were nearly identical (S1 Table), suggesting that HCF-1rep1 O-GlcNAcylation has minor effects on proteolysis and vice-versa. Fig 4A illustrates schematically the results of the O-GlcNAcylation analysis of the HCF-1rep1 uncleaved precursor protein. Because of the new trypsin cleavage sites, full peptide coverage of the HCF-1 sequence was achieved. Previously identified confident O-GlcNAcylation sites (red squares) in Region I [9] were confirmed. A cluster of four novel confident (red squares) and two novel potential (blue squares) O-GlcNAcylation sites in the Region II CEE, and one novel confident and two novel potential O-GlcNAcylation sites in Region III were identified. Thus, in summary, we conclude that the Region II CEE (i) enhances HCF-1PRO-repeat cleavage, (ii) associates efficiently with OGT, and (iii) contains a cluster of O-GlcNAcylated serines and threonines.


Distinct OGT-Binding Sites Promote HCF-1 Cleavage.

Bhuiyan T, Waridel P, Kapuria V, Zoete V, Herr W - PLoS ONE (2015)

Region II CEE O-GlcNAcylation and HCF-1PRO-repeat proteolysis are independent OGT activities.(A) (Left) The full-length (FL) HCF-1rep1 precursor (band a) and the N-terminal cleavage product (band b) were purified from HEK 293 lysates via α-HA-epitope immunoprecipitation and visualized by Coomassie staining. The bands were analyzed for O-GlcNAcylation and phosphorylation sites by LC-MS/MS. (Right) Schematic representation of identified HCF-1rep1 O-GlcNAcylation (squares) and phosphorylation (yellow circles) sites in the uncleaved HCF-1rep1 precursor. The HCF-1 sequences covered by the analysis (residues 867–1071) and the engineered trypsin cleavage sites A933K and M951K are indicated below the diagram. Red and blue squares indicate confident (Mascot score > 23 & probability of localization > 70%) and potential (Mascot score 14–22 or probability of localization 50–70%) O-GlcNAcylation sites, respectively. Squares surrounded in black indicate previously identified sites [9]. The HCF-1 Region II CEE amino acid sequence spanning a peptide sequence used in subsequent analyses (underlined: 901–933K) is shown below the diagram.(B) Analysis of a representative Region II CEE peptide (901–933K sequence shown in A) by LC-MS/MS for proportions of different O-GlcNAcylated forms. The proportions of 901–933K peptides containing 0, 1, 2 or 3 attached O-GlcNAc moieties are given for each sample in percent. HCF-1rep1 constructs were synthesized in HEK 293 cells and peptides were derived from constructs containing wild-type (WT) or mutated (E10A, E10D, E10Q, E10S, T17–22A) HCF-1PRO repeats, or containing a deletion of the HCF-1PRO-repeat sequence (∆PRO). The results with WT precursor, E10A, and E10S were confirmed in a second independent experiment.(C) HCF-1rep1 O-GlcNAcylation is not fundamental for HCF-1PRO-repeat cleavage. In vitro cleavage activities of wild-type OGT (WT) and an O-GlcNAcylation compromised OGT mutant (D554H_H558D) on selected HCF-1rep1 substrates. Cleavage and O-GlcNAcylation activities of constructs containing the full-length HCF-1rep1 sequence (FL), or the Region II CEE (+II) or Region III (+III) sequences were analyzed by immunoblot using the indicated antibodies. We note that the lack of the OGT D554H_H558D O-GlcNAcylation activity results in differential mobility of the HCF-1rep1 cleavage products during electrophoresis. Prominent (●) and faint (⭕) cleavage products are indicated.
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pone.0136636.g004: Region II CEE O-GlcNAcylation and HCF-1PRO-repeat proteolysis are independent OGT activities.(A) (Left) The full-length (FL) HCF-1rep1 precursor (band a) and the N-terminal cleavage product (band b) were purified from HEK 293 lysates via α-HA-epitope immunoprecipitation and visualized by Coomassie staining. The bands were analyzed for O-GlcNAcylation and phosphorylation sites by LC-MS/MS. (Right) Schematic representation of identified HCF-1rep1 O-GlcNAcylation (squares) and phosphorylation (yellow circles) sites in the uncleaved HCF-1rep1 precursor. The HCF-1 sequences covered by the analysis (residues 867–1071) and the engineered trypsin cleavage sites A933K and M951K are indicated below the diagram. Red and blue squares indicate confident (Mascot score > 23 & probability of localization > 70%) and potential (Mascot score 14–22 or probability of localization 50–70%) O-GlcNAcylation sites, respectively. Squares surrounded in black indicate previously identified sites [9]. The HCF-1 Region II CEE amino acid sequence spanning a peptide sequence used in subsequent analyses (underlined: 901–933K) is shown below the diagram.(B) Analysis of a representative Region II CEE peptide (901–933K sequence shown in A) by LC-MS/MS for proportions of different O-GlcNAcylated forms. The proportions of 901–933K peptides containing 0, 1, 2 or 3 attached O-GlcNAc moieties are given for each sample in percent. HCF-1rep1 constructs were synthesized in HEK 293 cells and peptides were derived from constructs containing wild-type (WT) or mutated (E10A, E10D, E10Q, E10S, T17–22A) HCF-1PRO repeats, or containing a deletion of the HCF-1PRO-repeat sequence (∆PRO). The results with WT precursor, E10A, and E10S were confirmed in a second independent experiment.(C) HCF-1rep1 O-GlcNAcylation is not fundamental for HCF-1PRO-repeat cleavage. In vitro cleavage activities of wild-type OGT (WT) and an O-GlcNAcylation compromised OGT mutant (D554H_H558D) on selected HCF-1rep1 substrates. Cleavage and O-GlcNAcylation activities of constructs containing the full-length HCF-1rep1 sequence (FL), or the Region II CEE (+II) or Region III (+III) sequences were analyzed by immunoblot using the indicated antibodies. We note that the lack of the OGT D554H_H558D O-GlcNAcylation activity results in differential mobility of the HCF-1rep1 cleavage products during electrophoresis. Prominent (●) and faint (⭕) cleavage products are indicated.
Mentions: We purified full-length HCF-1rep1 with the engineered trypsin cleavage sites after transient synthesis in HEK 293 cells and analyzed the uncleaved precursor band (Fig 4A, band a), as well as the N-terminal cleavage product (band b) for O-GlcNAcylation. The identified O-GlcNAcylation sites of the HCF-1rep1 precursor and the N-terminal cleavage product were nearly identical (S1 Table), suggesting that HCF-1rep1 O-GlcNAcylation has minor effects on proteolysis and vice-versa. Fig 4A illustrates schematically the results of the O-GlcNAcylation analysis of the HCF-1rep1 uncleaved precursor protein. Because of the new trypsin cleavage sites, full peptide coverage of the HCF-1 sequence was achieved. Previously identified confident O-GlcNAcylation sites (red squares) in Region I [9] were confirmed. A cluster of four novel confident (red squares) and two novel potential (blue squares) O-GlcNAcylation sites in the Region II CEE, and one novel confident and two novel potential O-GlcNAcylation sites in Region III were identified. Thus, in summary, we conclude that the Region II CEE (i) enhances HCF-1PRO-repeat cleavage, (ii) associates efficiently with OGT, and (iii) contains a cluster of O-GlcNAcylated serines and threonines.

Bottom Line: HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle.Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage.These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity.

View Article: PubMed Central - PubMed

Affiliation: Center for Integrative Genomics, University of Lausanne, Génopode, Lausanne, Switzerland.

ABSTRACT
Human HCF-1 (also referred to as HCFC-1) is a transcriptional co-regulator that undergoes a complex maturation process involving extensive O-GlcNAcylation and site-specific proteolysis. HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by O-GlcNAc transferase (OGT), which thus displays an unusual dual enzymatic activity. OGT cleaves HCF-1 at six highly conserved 26 amino acid repeat sequences called HCF-1PRO repeats. Here we characterize the substrate requirements for OGT cleavage of HCF-1. We show that the HCF-1PRO-repeat cleavage signal possesses particular OGT-binding properties. The glutamate residue at the cleavage site that is intimately involved in the cleavage reaction specifically inhibits association with OGT and its bound cofactor UDP-GlcNAc. Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage. These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity.

No MeSH data available.


Related in: MedlinePlus