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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

The glutamate residue at position 10 in the HCF-1PRO repeat inhibits OGT binding.(A) (Top) Schematic representation of the human HCF-1 protein showing the six HCF-1PRO repeats as yellow triangles. (Bottom) Schematic of the recombinant HCF-1rep1 precursor (HCF-1 residues 867–1071), containing the HCF-1PRO repeat 1 (rep1) and surrounding sequences fused to glutathione-S-transferase (GST). The 26 residues of rep1 are shown and the site of proteolysis is marked at the C/E sequence by a black arrowhead. The 20 amino acid core sequence [14] is shaded gray, and the cleavage and threonine regions are indicated. (B) E10 exhibits inhibitory contributions to OGT binding. Computational estimation of the contributions to the binding free-energy, ΔGbind, of single amino acid residues in the HCF-1PRO repeat (P7-T14) for the association with OGT. Negative and positive values correspond to favorable and unfavorable OGT-association contributions, respectively, in the in silico structural complex (based on the 4N3B structure [22]). (C) An E10 to alanine substitution enhances HCF-1PRO-repeat–OGT binding. In vitro HCF-1rep1–OGT binding assay in the presence of UDP-GlcNAc with HCF-1rep1 constructs containing individual alanine substitutions in the HCF-1PRO repeat (P7A-T14A). HCF-1rep1 binding to OGT was quantified from an immunoblot (S1B Fig) as ratio of OGT-bound HCF-1rep1 to total HCF-1rep1 in the assay. Obtained values are presented as log2 fold change relative to wild-type HCF-1rep1–OGT binding. (D) HCF-1PRO-repeat–OGT binding is influenced by the E10 side-chain properties and exhibits sensitivity to UDP-GlcNAc. In vitro HCF-1rep1–OGT binding assay. Constructs with wild-type (WT), E10 missense mutations (E10A, E10D, E10Q, and E10S), and T17-T22A mutations were incubated in the presence (left panel) or absence (right panel) of UDP-GlcNAc. Detection of OGT and HCF-1rep1 was performed using the indicated antibodies. *, IgG heavy chain. (E) Computational estimation of the contributions to the binding free-energy, ΔGbind, of single amino acid residues at the E10 cleavage site for the association with OGT. Shown are residue contributions of WT (E10), alanine (E10A), aspartate (E10D) or glutamine (E10Q) side-chains in the presence or absence of UDP-GlcNAc in the in silico structural complex (based on the 4N3B structure). (F) Close-up view (4N3C crystal structure [22]) of the OGT active site with the HCF-1PRO repeat and UDP-GlcNAc. The deprotonated E10 oxygen atom exhibits an unfavorable interaction with OGT. (G) Snapshot from a molecular dynamics simulation (based on the 4N3B structure) of the OGT active site in complex with the HCF-1PRO repeat without UDP-GlcNAc. The displayed frame is representative of the average distances sampled along the simulations. In (F) and (G), the E10 side-chain is shown in ball and stick representation (carbons: gray, nitrogen: blue, oxygens: red), and dashed lines indicate distances between atoms.
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pone.0136636.g001: The glutamate residue at position 10 in the HCF-1PRO repeat inhibits OGT binding.(A) (Top) Schematic representation of the human HCF-1 protein showing the six HCF-1PRO repeats as yellow triangles. (Bottom) Schematic of the recombinant HCF-1rep1 precursor (HCF-1 residues 867–1071), containing the HCF-1PRO repeat 1 (rep1) and surrounding sequences fused to glutathione-S-transferase (GST). The 26 residues of rep1 are shown and the site of proteolysis is marked at the C/E sequence by a black arrowhead. The 20 amino acid core sequence [14] is shaded gray, and the cleavage and threonine regions are indicated. (B) E10 exhibits inhibitory contributions to OGT binding. Computational estimation of the contributions to the binding free-energy, ΔGbind, of single amino acid residues in the HCF-1PRO repeat (P7-T14) for the association with OGT. Negative and positive values correspond to favorable and unfavorable OGT-association contributions, respectively, in the in silico structural complex (based on the 4N3B structure [22]). (C) An E10 to alanine substitution enhances HCF-1PRO-repeat–OGT binding. In vitro HCF-1rep1–OGT binding assay in the presence of UDP-GlcNAc with HCF-1rep1 constructs containing individual alanine substitutions in the HCF-1PRO repeat (P7A-T14A). HCF-1rep1 binding to OGT was quantified from an immunoblot (S1B Fig) as ratio of OGT-bound HCF-1rep1 to total HCF-1rep1 in the assay. Obtained values are presented as log2 fold change relative to wild-type HCF-1rep1–OGT binding. (D) HCF-1PRO-repeat–OGT binding is influenced by the E10 side-chain properties and exhibits sensitivity to UDP-GlcNAc. In vitro HCF-1rep1–OGT binding assay. Constructs with wild-type (WT), E10 missense mutations (E10A, E10D, E10Q, and E10S), and T17-T22A mutations were incubated in the presence (left panel) or absence (right panel) of UDP-GlcNAc. Detection of OGT and HCF-1rep1 was performed using the indicated antibodies. *, IgG heavy chain. (E) Computational estimation of the contributions to the binding free-energy, ΔGbind, of single amino acid residues at the E10 cleavage site for the association with OGT. Shown are residue contributions of WT (E10), alanine (E10A), aspartate (E10D) or glutamine (E10Q) side-chains in the presence or absence of UDP-GlcNAc in the in silico structural complex (based on the 4N3B structure). (F) Close-up view (4N3C crystal structure [22]) of the OGT active site with the HCF-1PRO repeat and UDP-GlcNAc. The deprotonated E10 oxygen atom exhibits an unfavorable interaction with OGT. (G) Snapshot from a molecular dynamics simulation (based on the 4N3B structure) of the OGT active site in complex with the HCF-1PRO repeat without UDP-GlcNAc. The displayed frame is representative of the average distances sampled along the simulations. In (F) and (G), the E10 side-chain is shown in ball and stick representation (carbons: gray, nitrogen: blue, oxygens: red), and dashed lines indicate distances between atoms.

Mentions: Previous in vivo and in vitro cleavage studies of the HCF-1PRO repeat embedded in a heterologous context (i.e., the Oct-1 transcription factor) have shown that the activities of the cleavage and threonine regions are sensitive to alanine substitutions [9, 13]. To probe the role of HCF-1PRO-repeat residues for cleavage in their natural sequence environment, we used a recombinant cleavage substrate called HCF-1rep1 [9]. Fig 1A illustrates the structure of human HCF-1 with the six HCF-1PRO repeats shown in yellow. The HCF-1rep1 substrate, shown below the full-length HCF-1 structure, contains the first HCF-1PRO repeat embedded into its HCF-1 context. We performed in vitro cleavage assays with bacterially purified human OGT and HCF-1rep1 substrates with residues surrounding the cleavage site (P7-T14) mutated to alanine. The results (S1A Fig) paralleled those of the previous Oct-1/HCF-1 hybrid studies [9, 13], although the HCF-1PRO-repeat residues T11, H12, and E13 exhibited little importance in this assay, leading us to refine the HCF-1PRO-repeat cleavage region to the sequence encompassing residues P7 to E10, as shown in Fig 1A.


Distinct OGT-Binding Sites Promote HCF-1 Cleavage.

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

The glutamate residue at position 10 in the HCF-1PRO repeat inhibits OGT binding.(A) (Top) Schematic representation of the human HCF-1 protein showing the six HCF-1PRO repeats as yellow triangles. (Bottom) Schematic of the recombinant HCF-1rep1 precursor (HCF-1 residues 867–1071), containing the HCF-1PRO repeat 1 (rep1) and surrounding sequences fused to glutathione-S-transferase (GST). The 26 residues of rep1 are shown and the site of proteolysis is marked at the C/E sequence by a black arrowhead. The 20 amino acid core sequence [14] is shaded gray, and the cleavage and threonine regions are indicated. (B) E10 exhibits inhibitory contributions to OGT binding. Computational estimation of the contributions to the binding free-energy, ΔGbind, of single amino acid residues in the HCF-1PRO repeat (P7-T14) for the association with OGT. Negative and positive values correspond to favorable and unfavorable OGT-association contributions, respectively, in the in silico structural complex (based on the 4N3B structure [22]). (C) An E10 to alanine substitution enhances HCF-1PRO-repeat–OGT binding. In vitro HCF-1rep1–OGT binding assay in the presence of UDP-GlcNAc with HCF-1rep1 constructs containing individual alanine substitutions in the HCF-1PRO repeat (P7A-T14A). HCF-1rep1 binding to OGT was quantified from an immunoblot (S1B Fig) as ratio of OGT-bound HCF-1rep1 to total HCF-1rep1 in the assay. Obtained values are presented as log2 fold change relative to wild-type HCF-1rep1–OGT binding. (D) HCF-1PRO-repeat–OGT binding is influenced by the E10 side-chain properties and exhibits sensitivity to UDP-GlcNAc. In vitro HCF-1rep1–OGT binding assay. Constructs with wild-type (WT), E10 missense mutations (E10A, E10D, E10Q, and E10S), and T17-T22A mutations were incubated in the presence (left panel) or absence (right panel) of UDP-GlcNAc. Detection of OGT and HCF-1rep1 was performed using the indicated antibodies. *, IgG heavy chain. (E) Computational estimation of the contributions to the binding free-energy, ΔGbind, of single amino acid residues at the E10 cleavage site for the association with OGT. Shown are residue contributions of WT (E10), alanine (E10A), aspartate (E10D) or glutamine (E10Q) side-chains in the presence or absence of UDP-GlcNAc in the in silico structural complex (based on the 4N3B structure). (F) Close-up view (4N3C crystal structure [22]) of the OGT active site with the HCF-1PRO repeat and UDP-GlcNAc. The deprotonated E10 oxygen atom exhibits an unfavorable interaction with OGT. (G) Snapshot from a molecular dynamics simulation (based on the 4N3B structure) of the OGT active site in complex with the HCF-1PRO repeat without UDP-GlcNAc. The displayed frame is representative of the average distances sampled along the simulations. In (F) and (G), the E10 side-chain is shown in ball and stick representation (carbons: gray, nitrogen: blue, oxygens: red), and dashed lines indicate distances between atoms.
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Related In: Results  -  Collection

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Show All Figures
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pone.0136636.g001: The glutamate residue at position 10 in the HCF-1PRO repeat inhibits OGT binding.(A) (Top) Schematic representation of the human HCF-1 protein showing the six HCF-1PRO repeats as yellow triangles. (Bottom) Schematic of the recombinant HCF-1rep1 precursor (HCF-1 residues 867–1071), containing the HCF-1PRO repeat 1 (rep1) and surrounding sequences fused to glutathione-S-transferase (GST). The 26 residues of rep1 are shown and the site of proteolysis is marked at the C/E sequence by a black arrowhead. The 20 amino acid core sequence [14] is shaded gray, and the cleavage and threonine regions are indicated. (B) E10 exhibits inhibitory contributions to OGT binding. Computational estimation of the contributions to the binding free-energy, ΔGbind, of single amino acid residues in the HCF-1PRO repeat (P7-T14) for the association with OGT. Negative and positive values correspond to favorable and unfavorable OGT-association contributions, respectively, in the in silico structural complex (based on the 4N3B structure [22]). (C) An E10 to alanine substitution enhances HCF-1PRO-repeat–OGT binding. In vitro HCF-1rep1–OGT binding assay in the presence of UDP-GlcNAc with HCF-1rep1 constructs containing individual alanine substitutions in the HCF-1PRO repeat (P7A-T14A). HCF-1rep1 binding to OGT was quantified from an immunoblot (S1B Fig) as ratio of OGT-bound HCF-1rep1 to total HCF-1rep1 in the assay. Obtained values are presented as log2 fold change relative to wild-type HCF-1rep1–OGT binding. (D) HCF-1PRO-repeat–OGT binding is influenced by the E10 side-chain properties and exhibits sensitivity to UDP-GlcNAc. In vitro HCF-1rep1–OGT binding assay. Constructs with wild-type (WT), E10 missense mutations (E10A, E10D, E10Q, and E10S), and T17-T22A mutations were incubated in the presence (left panel) or absence (right panel) of UDP-GlcNAc. Detection of OGT and HCF-1rep1 was performed using the indicated antibodies. *, IgG heavy chain. (E) Computational estimation of the contributions to the binding free-energy, ΔGbind, of single amino acid residues at the E10 cleavage site for the association with OGT. Shown are residue contributions of WT (E10), alanine (E10A), aspartate (E10D) or glutamine (E10Q) side-chains in the presence or absence of UDP-GlcNAc in the in silico structural complex (based on the 4N3B structure). (F) Close-up view (4N3C crystal structure [22]) of the OGT active site with the HCF-1PRO repeat and UDP-GlcNAc. The deprotonated E10 oxygen atom exhibits an unfavorable interaction with OGT. (G) Snapshot from a molecular dynamics simulation (based on the 4N3B structure) of the OGT active site in complex with the HCF-1PRO repeat without UDP-GlcNAc. The displayed frame is representative of the average distances sampled along the simulations. In (F) and (G), the E10 side-chain is shown in ball and stick representation (carbons: gray, nitrogen: blue, oxygens: red), and dashed lines indicate distances between atoms.
Mentions: Previous in vivo and in vitro cleavage studies of the HCF-1PRO repeat embedded in a heterologous context (i.e., the Oct-1 transcription factor) have shown that the activities of the cleavage and threonine regions are sensitive to alanine substitutions [9, 13]. To probe the role of HCF-1PRO-repeat residues for cleavage in their natural sequence environment, we used a recombinant cleavage substrate called HCF-1rep1 [9]. Fig 1A illustrates the structure of human HCF-1 with the six HCF-1PRO repeats shown in yellow. The HCF-1rep1 substrate, shown below the full-length HCF-1 structure, contains the first HCF-1PRO repeat embedded into its HCF-1 context. We performed in vitro cleavage assays with bacterially purified human OGT and HCF-1rep1 substrates with residues surrounding the cleavage site (P7-T14) mutated to alanine. The results (S1A Fig) paralleled those of the previous Oct-1/HCF-1 hybrid studies [9, 13], although the HCF-1PRO-repeat residues T11, H12, and E13 exhibited little importance in this assay, leading us to refine the HCF-1PRO-repeat cleavage region to the sequence encompassing residues P7 to E10, as shown in Fig 1A.

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