LiteFold/DisProt huggingface dataset:
Protein: DP03292 (UniProt P00742)
Region: residues 99	to 116
Annotation: disorder to order (Structural transition)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
AspH-TPR-Ox was successfully co-crystallized with a 39- residue fragment of hFX EGF1 (AspH-TPR-Ox:hFX, Fig. 3 and Supplementary Fig. 9). The AspH-TPR-Ox structure reveals electron density at the active site corresponding to 18 of the 39 hFX EGF1 substrate residues (aa 99–116: -GKC3KDGLGEYTC4TC5LEGF-) and for a disulfide linkage between Cys101hFX and Cys110hFX (Fig. 3e). Highly specific interactions, including multiple protein–protein/peptide interactions with both AspH-Ox and TPR domains are apparent (Fig. 3b–d). The N-terminal region of hFX (aa 100–105) interacts with the AspH-Ox domain and the C-terminal region of hFX (aa 106–116) interacts with the TPR domain
Cross-references: PDB:5JZU
PDB:5JQY
PDB:5JZ8
PDB:6RK9
Protein: DP03292 (UniProt P00742)
Region: residues 106	to 116
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Highly specific interactions, including multiple protein–protein/peptide interactions with both AspH-Ox and TPR domains are apparent (Fig. 3b–d). The N-terminal region of hFX (aa 100–105) interacts with the AspH-Ox domain and the C-terminal region of hFX (aa 106–116) interacts with the TPR domain
Cross-references: PDB:5JZU
PDB:5JQY
PDB:5JZ8
PDB:6RK9
Protein: DP03292 (UniProt P00742)
Region: residues 100	to 105
Annotation: protein binding (Molecular function)
Evidence: x-ray crystallography evidence used in manual assertion
Supporting statements:
Highly specific interactions, including multiple protein–protein/peptide interactions with both AspH-Ox and TPR domains are apparent (Fig. 3b–d). The N-terminal region of hFX (aa 100–105) interacts with the AspH-Ox domain and the C-terminal region of hFX (aa 106–116) interacts with the TPR domain
Cross-references: PDB:5JZU
PDB:5JQY
PDB:5JZ8
PDB:6RK9
Protein: DP03293 (UniProt O43791)
Region: residues 357	to 374
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
No electron density was observed for the residues comprising the predicted nuclear localization signal (residues 360–374) and BTB5–6 (residues 270–295), and they were not built in the model;
Cross-references: PDB:4J8Z
Protein: DP03293 (UniProt O43791)
Region: residues 169	to 178
Annotation: flexible linker (Disorder function)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
No electron density was observed for residues 169–176 and 357–374 of SPOP169–374 Y353E, and residues comprising the 3–4loop were associated with higher crystallographic B factors, as reported previously (Zhuanget  al., 2009; Erringtonet  al.,2012).
Human SPOP is a 374-residue protein that contains an N-terminal MATH domain (residues 28–166) that recruits substrates and a central BTB domain (residues 177–296) that mediates dimerization and interactions with Cul3 (Fig. 1a).
Cross-references: PDB:4J8Z
Protein: DP03293 (UniProt O43791)
Region: residues 169	to 178
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
No electron density was observed for residues 169–176 and 357–374 of SPOP169–374 Y353E, and residues comprising the 3–4loop were associated with higher crystallographic B factors, as reported previously (Zhuanget  al., 2009; Erringtonet  al.,2012).
Cross-references: PDB:4J8Z
Protein: DP03293 (UniProt O43791)
Region: residues 361	to 374
Annotation: localization (Biological process)
Evidence: fluorescence microscopy evidence used in manual assertion
Supporting statements:
Co-immunoprecipitation experiments in SW1990 cells showed that SPOP interacted with the stem-cell marker NANOG, and this interaction has recently been shown to play a critical role in regulating progression of prostate cancer. We showed that, in one patient with pancreatic cancer, the expression of a truncated form of SPOP (p.Q360*) lacking the nuclear localization signal led to nuclear accumulation of NANOG, which promoted growth and metastasis of pancreatic cancer cells. Our results suggest that SPOP suppresses progression of pancreatic cancer by promoting the ubiquitination and subsequent degradation of NANOG. These results identify the SPOP-NANOG interaction as a potential therapeutic target against pancreatic cancer.
Since the Q360* mutation is located in the linker between the BTB domain and the nuclear localization sequence, we speculated that the mutated SPOP sequence containing the C1078T mutation encodes a truncated protein without the nuclear localization sequence (Supplementary Fig. 1a-c). Indeed, fluorescence microscopy and nuclear/cytoplasmic separation showed that SPOP-WT mainly located in cell nucleus, SPOP-Q360* mainly located in cell cytoplasm, NANOG localized predominantly in the nucleus (Fig. 7b, c).
The inability of SPOP-Q360* to accumulate in the nucleus was associated with an inability to bind importin subunit alpha-6 and 7 (IPOA6 and IPOA7), a shuttle carrier between nucleus and cytoplasm (Supplementary Fig. 2).
The patient-derived Q360* mutation disrupts nuclear localization of SPOP and impairs the SPOP-mediated poly-ubiquitination and degradation of NANOG.
These results suggest that the Q360* mutation alters SPOP localization and allows hyperaccumulation of NANOG in the nucleus, where it can promote cell proliferation.
Protein: DP03293 (UniProt O43791)
Region: residues 361	to 374
Annotation: molecular function regulator (Molecular function)
Evidence: qualitative western immunoblotting evidence used in manual assertion
Supporting statements:
Co-immunoprecipitation experiments in SW1990 cells showed that SPOP interacted with the stem-cell marker NANOG, and this interaction has recently been shown to play a critical role in regulating progression of prostate cancer. We showed that, in one patient with pancreatic cancer, the expression of a truncated form of SPOP (p.Q360*) lacking the nuclear localization signal led to nuclear accumulation of NANOG, which promoted growth and metastasis of pancreatic cancer cells. Our results suggest that SPOP suppresses progression of pancreatic cancer by promoting the ubiquitination and subsequent degradation of NANOG. These results identify the SPOP-NANOG interaction as a potential therapeutic target against pancreatic cancer.
SPOP-Q360* repressed levels of NANOG and other downstream proteins to a much weaker extent than SPOP-WT (Fig. 7d), and these results were associated with impaired interaction between SPOP and NANOG, as well as impaired ubiquitination of NANOG (Fig. 7e, f).
The patient-derived Q360* mutation disrupts nuclear localization of SPOP and impairs the SPOP-mediated poly-ubiquitination and degradation of NANOG.
These results suggest that the Q360* mutation alters SPOP localization and allows hyperaccumulation of NANOG in the nucleus, where it can promote cell proliferation.
Protein: DP03293 (UniProt O43791)
Region: residues 361	to 374
Annotation: molecular function inhibitor activity (Molecular function)
Evidence: experimental phenotypic evidence used in manual assertion
Supporting statements:
Co-immunoprecipitation experiments in SW1990 cells showed that SPOP interacted with the stem-cell marker NANOG, and this interaction has recently been shown to play a critical role in regulating progression of prostate cancer. We showed that, in one patient with pancreatic cancer, the expression of a truncated form of SPOP (p.Q360*) lacking the nuclear localization signal led to nuclear accumulation of NANOG, which promoted growth and metastasis of pancreatic cancer cells. Our results suggest that SPOP suppresses progression of pancreatic cancer by promoting the ubiquitination and subsequent degradation of NANOG. These results identify the SPOP-NANOG interaction as a potential therapeutic target against pancreatic cancer.
To examine how the patient-derived Q360* mutation may disrupt the tumor-suppressive role of SPOP in pancreatic cancer, we transiently upregulated expression of SPOP-WT or SPOP-Q360* in cells overexpressing NANOG. We found that the Q360* mutation weakened the antiproliferative effect of SPOP under these conditions (Fig. 7a).
Protein: DP03294 (UniProt P02760)
Region: residues 340	to 352
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
There was no electron density for residues 1 to 24 or 135 to 145. The C-terminal residues are probably disordered.
Region 135-147 is not visible in the structure, which corresponds to region 340-352 of the amino acid sequence. Protein AMBP is proteolitically cleaved into separately functioning proteins. This region is part of protein bikunin.
Cross-references: PDB:1BIK
Protein: DP03294 (UniProt P02760)
Region: residues 206	to 229
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
There was no electron density for residues 1 to 24 or 135 to 145.
Bikunin has two glycosylation sites, residues Ser10 and Asn45. Ser10 is the more heavily glycosylated (Hochstrasser et al., 1981) but it is in a section of peptide that is not visible in our maps.
The IDR characterized in the publication and spanning residues 1-24 corresponds to region 206-229 of the amino acid sequence. Protein AMBP is proteolitically cleaved into separately functioning proteins. This region is part of protein bikunin.
Cross-references: PDB:1BIK
Protein: DP03294 (UniProt P02760)
Region: residues 191	to 202
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
The first seven N-terminal residues, Gly1–Pro7, and the C-terminal residues Gly172–Lys193, as well as the Strep-tag II were not visible in the electron density map and hence were omitted from the final model.
Region 172-183 of the protein is not visible in the structure, which corresponds to region 191-202 of the associated UniProt sequence. Protein AMBP is proteolitically cleaved into separately functioning proteins. This region is part of protein α1-microglobulin.
Cross-references: PDB:3QKG
Protein: DP03294 (UniProt P02760)
Region: residues 213	to 217
Annotation: carbohydrate binding (Molecular function)
Evidence: mass spectrometry evidence used in manual assertion
Supporting statements:
The chondroitin sulphate chain is bound at its reducing end by the typical linkage structure Gal-Gal-Xyl to the Ser10 of bikunin whereas the C-terminal Asp of the heavy chain H3 esterifies C6 of an internal N-acetylgalactosamine inside the GAG chain.
Fractions 4 and 5 correspond to the peptides Va12-Gln14 and Alal-Gln14 of bikunin, respectively (Table 2). As expected [l, 141], Ser10 was recovered with a low yield during Edman degradation: it constitutes the attachment point of the chondroitin sulphate chain as shown by the monosaccharide analysis of fraction 4 (Xyl/Gal = 1:2.1).
A molecular ion at mlz 1900 was identified in fraction 4 (data not shown). The expected mlz for the bikunin-derived peptide is 1286. The difference (614Da) was explained by the presence of the tetrasaccharide Xyl-Gal-Gal-dehydrohexuronic acid, typical of a chondroitin AC lyase digest of Ser-linked GAG [16].
Ser10 corresponds to Ser215, since protein AMBP is proteolitically cleaved into separately functioning proteins and this region is part of protein bikunin (206-352).
Protein: DP03295 (UniProt Q92854)
Region: residues 649	to 677
Annotation: disorder (Structural state)
Evidence: X-ray crystallography-based structural model with missing residue coordinates used in manual assertion
Supporting statements:
Residues 1-2, 201-204 and 628-657 plus the His-tag are disordered in both subunits of the homodimer.
Residues 628-657 correspond to residues 649 and 677 of the original protein sequence as the first 21 residues are omitted (signal peptide)
Cross-references: PDB:1OLZ
Protein: DP03296 (UniProt P60484)
Region: residues 354	to 403
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Proteolytic digestion indicated that PTEN has unstructured or loosely folded regions of 7 and 49 residues at the N and C termini, respectively, and of 24 residues in an internal loop (residues 286–309; Figure 1B; data not shown).
Protein: DP03296 (UniProt P60484)
Region: residues 353	to 403
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Secondary structure prediction and the results of proteolytic digestion experiments suggest that the tail is a relatively unstructured and presumably flexible region.
Protein: DP03296 (UniProt P60484)
Region: residues 353	to 403
Annotation: molecular function regulator (Molecular function)
Evidence: deletion mutation phenotypic evidence used in manual assertion
Supporting statements:
Here, we show that the PTEN tail is necessary for maintaining protein stability and that it also acts to inhibit PTEN function. Thus, removing the tail results in a loss of stability but does not result in a loss of function because the resultant protein is more active. Furthermore, tail-dependent regulation of stability and activity is linked to the phosphorylation of three residues (S380, T382, and T383) within the tail. Therefore, the tail is likely to mediate the regulation of PTEN function through phosphorylation.
The PTEN tail modulates PTEN stability.
In keeping with these results, it was recently reported that the steady-state level of PTEN;1-351 is reduced compared to that of PTEN;WT when the protein is produced by transfection in COS-7 cells (14). Together these data suggest that the PTEN tail, while not required for the functional activity of the protein, is required for maintaining stability.
The tail domain modulates PTEN biological activity.
Surprisingly, at equivalent input plasmid concentrations PTEN;1-353 reproducibly induced a greater increase in G1 than PTEN;WT (data not shown). Next, the activities of PTEN;WT and PTEN;1-353 were compared when the proteins were produced at similar steady-state levels. Plasmid titration indicated that equivalent protein levels were obtained at 2 μg of PTEN;1-353 and 0.5 μg of PTEN;WT (Fig. ​(Fig.2A2A and B). At these levels PTEN;1-353 induced a significantly more robust G1 arrest (Fig. ​(Fig.2C).2C).
We next compared PTEN;WT and PTEN;1-353 in a FKHR transcriptional activation assay.
We next compared PTEN;WT and PTEN;1-353 in a FKHR transcriptional activation assay. Consistent with the results obtained in the cell cycle assay (Fig. ​(Fig.2C),2C), the ability of PTEN;1-353 to induce FKHR transcriptional activity was enhanced compared to that of PTEN;WT. Furthermore, at every DNA plasmid concentration tested, PTEN;1-353 induced FKHR activation more efficiently than PTEN;WT, although protein levels were reduced by more than fourfold. These results suggest that the PTEN tail not only plays a role in maintaining its protein stability but also in regulating its biological activity. Specifically, these data suggest that the tail acts to restrict or inhibit PTEN function.
Protein: DP03296 (UniProt P60484)
Region: residues 378	to 382
Annotation: phosphorylation display site (Disorder function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Furthermore, tail-dependent regulation of stability and activity is linked to the phosphorylation of three residues (S380, T382, and T383) within the tail. Therefore, the tail is likely to mediate the regulation of PTEN function through phosphorylation.
PTEN is phosphorylated within the tail domain.
Next, every serine and threonine in the tail was mutated either singly or in clusters to alanine. These PTEN mutants were transfected into U2-OS cells and labeled with orthophosphate. No single-amino-acid substitution abrogated or significantly reduced the total phosphorylation of PTEN (data not shown). However, the substitution of a serine/threonine cluster, S380, T382, T383, and S385 (the A4 mutant), did significantly alter total PTEN phosphorylation.
These results indicate that most, if not all, of the PTEN tail phosphorylation occurs on serine 370 and one or more sites of the A4 cluster (S380, T382, T383, and S385).
Mutation of the phosphorylation sites in the tail alter PTEN stability and biological activity.
Taken together, these data show that the increased activity associated with deletion of the tail is entirely mimicked by mutations within the A4 cluster, specifically S380, T382, or T383.
The above data raised the possibility that phosphorylation of these three specific residues (S380, T382, and T383) might be required to maintain PTEN in a stable yet relatively inactive state.
In keeping with the data for PTEN;A4 and for the individual phosphorylation site mutants (with mutations S380A, T382A, and T383A), PTEN;A3 was found to have a reduced protein half-life, to be expressed at lower steady-state levels, and to be more active than wild-type PTEN in biological assays (Fig. ​(Fig.6C to F).
This region is associated to S380.
Protein: DP03296 (UniProt P60484)
Region: residues 380	to 384
Annotation: phosphorylation display site (Disorder function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Furthermore, tail-dependent regulation of stability and activity is linked to the phosphorylation of three residues (S380, T382, and T383) within the tail. Therefore, the tail is likely to mediate the regulation of PTEN function through phosphorylation.
PTEN is phosphorylated within the tail domain.
Next, every serine and threonine in the tail was mutated either singly or in clusters to alanine. These PTEN mutants were transfected into U2-OS cells and labeled with orthophosphate. No single-amino-acid substitution abrogated or significantly reduced the total phosphorylation of PTEN (data not shown). However, the substitution of a serine/threonine cluster, S380, T382, T383, and S385 (the A4 mutant), did significantly alter total PTEN phosphorylation.
These results indicate that most, if not all, of the PTEN tail phosphorylation occurs on serine 370 and one or more sites of the A4 cluster (S380, T382, T383, and S385).
Mutation of the phosphorylation sites in the tail alter PTEN stability and biological activity.
Taken together, these data show that the increased activity associated with deletion of the tail is entirely mimicked by mutations within the A4 cluster, specifically S380, T382, or T383.
The above data raised the possibility that phosphorylation of these three specific residues (S380, T382, and T383) might be required to maintain PTEN in a stable yet relatively inactive state.
In keeping with the data for PTEN;A4 and for the individual phosphorylation site mutants (with mutations S380A, T382A, and T383A), PTEN;A3 was found to have a reduced protein half-life, to be expressed at lower steady-state levels, and to be more active than wild-type PTEN in biological assays (Fig. ​(Fig.6C to F).
This region is associated to T382.
Protein: DP03296 (UniProt P60484)
Region: residues 381	to 385
Annotation: phosphorylation display site (Disorder function)
Evidence: mutant phenotype evidence used in manual assertion
Supporting statements:
Furthermore, tail-dependent regulation of stability and activity is linked to the phosphorylation of three residues (S380, T382, and T383) within the tail. Therefore, the tail is likely to mediate the regulation of PTEN function through phosphorylation.
PTEN is phosphorylated within the tail domain.
Next, every serine and threonine in the tail was mutated either singly or in clusters to alanine. These PTEN mutants were transfected into U2-OS cells and labeled with orthophosphate. No single-amino-acid substitution abrogated or significantly reduced the total phosphorylation of PTEN (data not shown). However, the substitution of a serine/threonine cluster, S380, T382, T383, and S385 (the A4 mutant), did significantly alter total PTEN phosphorylation.
These results indicate that most, if not all, of the PTEN tail phosphorylation occurs on serine 370 and one or more sites of the A4 cluster (S380, T382, T383, and S385).
Mutation of the phosphorylation sites in the tail alter PTEN stability and biological activity.
Taken together, these data show that the increased activity associated with deletion of the tail is entirely mimicked by mutations within the A4 cluster, specifically S380, T382, or T383.
The above data raised the possibility that phosphorylation of these three specific residues (S380, T382, and T383) might be required to maintain PTEN in a stable yet relatively inactive state.
In keeping with the data for PTEN;A4 and for the individual phosphorylation site mutants (with mutations S380A, T382A, and T383A), PTEN;A3 was found to have a reduced protein half-life, to be expressed at lower steady-state levels, and to be more active than wild-type PTEN in biological assays (Fig. ​(Fig.6C to F).
This region is associated to T383.
Protein: DP03296 (UniProt P60484)
Region: residues 286	to 309
Annotation: disorder (Structural state)
Evidence: cleavage assay evidence used in manual assertion
Supporting statements:
Proteolytic digestion indicated that PTEN has unstructured or loosely folded regions of 7 and 49 residues at the N and C termini, respectively, and of 24 residues in an internal loop (residues 286–309; Figure 1B; data not shown).
Protein: DP03296 (UniProt P60484)
Region: residues 366	to 385
Annotation: self-inhibition (Disorder function)
Evidence: phosphatase assay evidence used in manual assertion
Supporting statements:
Phosphorylation of six residues in the C-terminal tail of PTEN results in auto-inhibitory interactions with the phosphatase and C2 domains, effectively blocking both the active site and the membrane-binding interface of PTEN.
Phosphorylation of the C-terminal tail differentially inhibits PTEN towards distinct substrates
To compare the activities and specificities of differentially phosphorylated PTEN constructs, phosphatase assays were conducted against four substrates: a phosphotyrosine-containing acidic polypeptide, the soluble lipid diC8-PIP3, inositol tetrakis phosphate (IP4) and liposome-incorporated PIP3 (Figures 1B–1E).
We found that phosphorylation of PTEN caused a dramatic reduction in activity against all substrates. Intriguingly, when PTEN was phosphorylated on only four sites (pSer380/pThr382/pThr383/pSer385) by using the phosphorylated PTEN-2A mutant (Phos-PTEN-2A), activity against the phosphopeptide and soluble diC8-PIP3/IP4 substrates was restored (Figures 1B–1D), but PTEN remained inhibited against liposome-incorporated PIP3 substrate (Figure 1E; Supplementary Figure S3). This suggests that pThr366 and pSer370 may have a role in occluding the active site of PTEN, whereas pSer380, pThr382, pThr383 and pSer385 have a role in occluding the membrane-binding surface. Phos-PTEN-4A, phosphorylated on only two sites, pThr366 and pSer370, showed similar activities with all substrates to that of dephosphorylated PTEN-4A, with only a slight reduction in activity against liposome-incorporated PIP3
Region inside the disordered C-terminal tail that include multiple phosphorylation sites: T366, S370, S380, T382, T383, S385