Limits...
Mining the TRAF6/p62 interactome for a selective ubiquitination motif.

Jadhav TS, Wooten MW, Wooten MC - BMC Proc (2011)

Bottom Line: NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination.Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination.The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cellular and Molecular Biosciences, Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, AL, 36849, USA. wootemc@auburn.edu.

ABSTRACT
A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [-(hydrophobic)-k-(hydrophobic)-x-x-(hydrophobic)- (polar)-(hydrophobic)-(polar)-(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

No MeSH data available.


Structural context of predicted TRAF6/p62 ubiquitination sites within 30 proteins containing perfect motif matches. A: Distribution of perfect match motif sites based on secondary structure for the experimental and negative datasets. B: Percentage distribution of predicted sites in disordered protein regions and domain structures. C: Relative proportions of perfect match sites found in three protein regions, N-terminus, Middle and C-terminus. D: Identification of perfect match Lysines as likely occupying exposed or buried protein regions as determined by solvent accessibility predictions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3090762&req=5

Figure 5: Structural context of predicted TRAF6/p62 ubiquitination sites within 30 proteins containing perfect motif matches. A: Distribution of perfect match motif sites based on secondary structure for the experimental and negative datasets. B: Percentage distribution of predicted sites in disordered protein regions and domain structures. C: Relative proportions of perfect match sites found in three protein regions, N-terminus, Middle and C-terminus. D: Identification of perfect match Lysines as likely occupying exposed or buried protein regions as determined by solvent accessibility predictions.

Mentions: We further sought to incorporate sequence information as well as information from sequence derived structural features of these proteins into the validation process. To do so, four potential structural features of the predicted high probability sites were evaluated: secondary structure, relative distribution within the protein, solvent accessibility, and the intrinsic disorder within the protein domain. Structural analysis was conducted using secondary structure, protein domain, and disorder prediction algorithms [25]. Our results indicated that approximately one-half of the ubiquitination sites were predicted to be in loops (Figure 5A) and disordered regions (Figure 5B). Beta-sheets had the least representation of predicted ubiquitination sites (with 15% sites in experimental and none from negative datasets). The predicted ubiquitinated site was found at a significantly greater rate in the loop regions than in the beta sheets of the protein structure (P = 0.0001). The second most common secondary structure was an alpha-helix (Figure 5A). Alpha helices and loops are usually found on the surface of proteins and are tend to easily accessible for post-translational modifications. The predicted sites showed significantly high occurrence of sites in helices and loops as compared to beta sheets (P = 0.0001). This was in agreement with previously reported findings on preferred in vivo ubiquitination sites in yeast proteins [28]. As an example where surface accessibility has been shown to define a ubiquitination site, position of Lysine 507 of Smad4 is ubiquitinated in the fully solvent-accessible L3 loop with its side chain protruding from the L3 loop surface to the neighboring space [29].


Mining the TRAF6/p62 interactome for a selective ubiquitination motif.

Jadhav TS, Wooten MW, Wooten MC - BMC Proc (2011)

Structural context of predicted TRAF6/p62 ubiquitination sites within 30 proteins containing perfect motif matches. A: Distribution of perfect match motif sites based on secondary structure for the experimental and negative datasets. B: Percentage distribution of predicted sites in disordered protein regions and domain structures. C: Relative proportions of perfect match sites found in three protein regions, N-terminus, Middle and C-terminus. D: Identification of perfect match Lysines as likely occupying exposed or buried protein regions as determined by solvent accessibility predictions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3090762&req=5

Figure 5: Structural context of predicted TRAF6/p62 ubiquitination sites within 30 proteins containing perfect motif matches. A: Distribution of perfect match motif sites based on secondary structure for the experimental and negative datasets. B: Percentage distribution of predicted sites in disordered protein regions and domain structures. C: Relative proportions of perfect match sites found in three protein regions, N-terminus, Middle and C-terminus. D: Identification of perfect match Lysines as likely occupying exposed or buried protein regions as determined by solvent accessibility predictions.
Mentions: We further sought to incorporate sequence information as well as information from sequence derived structural features of these proteins into the validation process. To do so, four potential structural features of the predicted high probability sites were evaluated: secondary structure, relative distribution within the protein, solvent accessibility, and the intrinsic disorder within the protein domain. Structural analysis was conducted using secondary structure, protein domain, and disorder prediction algorithms [25]. Our results indicated that approximately one-half of the ubiquitination sites were predicted to be in loops (Figure 5A) and disordered regions (Figure 5B). Beta-sheets had the least representation of predicted ubiquitination sites (with 15% sites in experimental and none from negative datasets). The predicted ubiquitinated site was found at a significantly greater rate in the loop regions than in the beta sheets of the protein structure (P = 0.0001). The second most common secondary structure was an alpha-helix (Figure 5A). Alpha helices and loops are usually found on the surface of proteins and are tend to easily accessible for post-translational modifications. The predicted sites showed significantly high occurrence of sites in helices and loops as compared to beta sheets (P = 0.0001). This was in agreement with previously reported findings on preferred in vivo ubiquitination sites in yeast proteins [28]. As an example where surface accessibility has been shown to define a ubiquitination site, position of Lysine 507 of Smad4 is ubiquitinated in the fully solvent-accessible L3 loop with its side chain protruding from the L3 loop surface to the neighboring space [29].

Bottom Line: NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination.Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination.The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

View Article: PubMed Central - HTML - PubMed

Affiliation: Program in Cellular and Molecular Biosciences, Department of Biological Sciences, 331 Funchess Hall, Auburn University, Auburn, AL, 36849, USA. wootemc@auburn.edu.

ABSTRACT
A new approach is described here to predict ubiquitinated substrates of the E3 ubiquitin ligase, TRAF6, which takes into account its interaction with the scaffold protein SQSTM1/p62. A novel TRAF6 ubiquitination motif defined as [-(hydrophobic)-k-(hydrophobic)-x-x-(hydrophobic)- (polar)-(hydrophobic)-(polar)-(hydrophobic)] was identified and used to screen the TRAF6/p62 interactome composed of 155 proteins, that were either TRAF6 or p62 interactors, or a negative dataset, composed of 54 proteins with no known association to either TRAF6 or p62. NRIF (K19), TrkA (K485), TrkB (K811), TrkC (K602 and K815), NTRK2 (K828), NTRK3 (K829) and MBP (K169) were found to possess a perfect match for the amino acid consensus motif for TRAF6/p62 ubiquitination. Subsequent analyses revealed that this motif was biased to the C-terminal regions of the protein (nearly 50% the sites), and had preference for loops (~50%) and helices (~37%) over beta-strands (15% or less). In addition, the motif was observed to be in regions that were highly solvent accessible (nearly 90%). Our findings suggest that specific Lysines may be selected for ubiquitination based upon an embedded code defined by a specific amino acid motif with structural determinants. Collectively, our results reveal an unappreciated role for the scaffold protein in targeting ubiquitination. The findings described herein could be used to aid in identification of other E3/scaffold ubiquitination sites.

No MeSH data available.