PrionDB: Extraction of mutation data from the literature

2005, PrionDB.

This data was extracted from Medline abstracts and full texts (when available) in an automated manner.

The table below describes different point mutations at a given position and provides links to other documents. The sentence(s) where the point mutations in PRIO_MOUSE at position 187 were found are listed after the table.

Point mutations at position T187 in PRIO_MOUSE

Protein	PRIO_MOUSE (P04925) Gene: Prnp,Prn-p (other point mutations)	Swiss-Prot Cross-reference table Family page
Position	T187
General numbering (PrionDB)	-
Domain	Not determined
Family alignments	Mammalian prion proteins Prion proteins (PRP, PRNP)
Other point mutations at the same position	Position 188 in Mammalian prion proteins family Position 188 in Prion proteins (PRP, PRNP) family
Reference #1	Lorenz H, Windl O, Kretzschmar HA J Biol Chem 2002 Mar 8;277(10):8508-16.	Medline
Text source	HTML and PDF full texts
Point mutation	T187K (True positive)
Point mutation	T187R (True positive)

Relevant sentences

Reference #1 (Lorenz H et al.): T187K

P101L (primers 1 and 2) , W144 Stop (primers 3 and 4) , Q159 Stop (primers 5 and 6) , D177N (primers 7 and 8) , T187K (primers 9 and 10) , T187R (primers 11 and 12) , and E199K (primers 13 and 14) mutations in the mouse prion protein open reading frame were obtained by a PCR-based site-directed mutagenesis method (14 ) with pBs-Prnp as template using the following primers: 5'-TCAGCAAACCAAAAACC-3' (primer 1) , 5'-GCTTGTTCCACTGATTA-3' (primer 2) , 5'-CCATTTTGGCAACGACTAGGAGGACCGCTACTACCG-3' (primer 3) , 5'-ATCATGGGCCTGCTCACGGCGCTCCCC-3' (primer 4) , 5'-GTACCGCTACCCTAACTAAGTGTACTACAGGCC-3' (primer 5) , 5'-ATGTTTTCACGGTAGTAGCGGTCCTCCCAGTCG-3' (primer 6) 5'-AACTGCGTCAATATCACC-3' (primer 7) , 5'-GTGCACGAAGTTGTTCTG-3' (primer 8) , 5'-CCATCAAGCAGCACAAGGTCACCACCACCACC-3' (primer 9) , 5'-TGATATTGACGCAGTCGTGCACGAAG-3' (primer 10) , 5'-GGGTCACCACCACCACCAAG-3' (primer 11) , 5'-TGTGCTGCTTGATGGTGATATTG-3' (primer 12) , 5'-AAGACCGATGTGAAGATG-3' (primer 13) , 5'-GGTGAAGTTCTCCCCCT-3' (primer 14)
Among these PrP mutants , there were three , GFP-PrP Q159 Stop , GFP-PrP T187K , and GFP-PrP T187R (corresponding to human PrP Q160 Stop , T188K , T188R) , that have so far never been cellularly examined and which were assayed in this study most intensively
After expression in N2a cells , the chimeric PrP missense mutants , i.e. GFP-PrP P101L , GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K , were fully glycosylated and synthesized to molecular masses of about 48-58 kDa like the chimera with wt PrP (Fig. 2 A)
Lane 1 , GFP-wtPrP ; lanes 2-8 , GFP-PrP mutants with amino acid substitutions P101L (lane 2) , W144 Stop (W144X , lane 3) , Q159 Stop (Q159X , lane 4) , D177N (lane 5) , T187K (lane 6) , T187R (lane 7) , E199K (lane 8)
Lanes 1 and 2 , untreated (-) and treated (+) GFP-wtPrP ; Lanes 3-7 , treated GFP-PrP mutants with amino acid substitutions P101L (lane 3) , D177N (lane 4) , T187K (lane 5) , T187R (lane 6) , and E199K (lane 7)
Single confocal sections of cells expressing GFP-wtPrP , GFP-PrP P101L , GFP-PrP W144 Stop (W144X) , GFP-PrP Q159 Stop (Q159X , from left to right , upper panel) and GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K (from left to right , lower panel) 48 h post-transfection
The microscopic examination revealed that the chimeric mutant proteins GFP-PrP P101L , GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K showed no difference in localization from GFP-wtPrP (Fig. 2 C)
Cellular Trafficking of the Chimeric PrP Mutants-- The three chimeric PrP mutants GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP Q159 Stop were further analyzed with respect to their cellular transport
This BFA-induced phenotype was also detected in N2a cells expressing GFP-PrP T187K and GFP-PrP T187R (Fig. 3 B) but was never observed for GFP-PrP Q159 Stop-expressing cells (Fig. 3 A)
Transfected cells showed the redistribution of GFP-PrP T187K and GFP-PrP T187R into the ER in the presence of BFA and a reorganization of discrete fluorescent signals or Golgi signals , respectively , in the absence of BFA , which clearly indicates a secretory pathway along the ER / Golgi complex for these two chimeric PrP mutants (Fig. 3 B)
The chimeric mutant PrPs T187K and T187R reveal a BFA-sensitive cellular phenotype like wt PrP , whereas chimeric PrP Q159 Stop (Q159X) localization is not influenced by BFA-incubation
B , signal (black) distributions of cells expressing (from left to right) GFP-wtPrP , GFP-PrP Q159 Stop (Q159X) , GFP-PrP T187K , GFP-PrP T187R
During PIPLC treatment for up to 40 min , the GFP-wtPrP- , GFP-PrP T187K- and GFP-PrP T187R-expressing cells showed an ongoing disappearance of the fluorescent signals on the cell surface , whereas no loss of fluorescence was observed for cells expressing GFP-PrP Q159 Stop (Fig. 4 )
Chimeric wt PrP and mutant PrP T187K and T187R (first , third , and fourth panel) could be cleaved off from the cell surface by incubation of the cells with PIPLC (1 unit / ml) for 40 min at 37 蚓
Western blotting revealed that the expression in N2a cells yielded glycosylated wt PrP , PrP T187K , and PrP T187R with molecular masses of ~27-37 kDa (Fig. 5 A)
A , N2a cells expressing wt PrP , PrP W144 Stop (W144X) , PrP Q159 Stop (Q159X) , PrP T187K , and PrP T187R (lanes 1-5) were extracted 48 h post-transfection , and equal amounts of total protein (100 痢) were subjected to Western blot analysis with anti-PrP antiserum Kan72
B , amount of secreted PrP in N2a cells expressing wt PrP , PrP W144 Stop (W144X) , PrP Q159 Stop (Q159X) , PrP T187K , and PrP T187R (lanes 1-5)
Under these conditions , secretion into the medium could easily be detected for wt PrP , PrP T187K , and PrP T187R but not for PrP W144 Stop and PrP Q159 Stop (Fig. 5 B) , supporting our data that the PrP nonsense mutants were strictly restricted to intracellular regions and , in contrast to the other examined PrPs , not trafficked along a secretory pathway
Biochemical Properties of the PrP Mutants-- To investigate whether PrP T187K and PrP T187R also exhibit an enhanced PK resistance like many other disease-associated full-length PrP mutants (7 , 8 ) or their chimeric PrP homologues , respectively (see Expression of Chimeric Prp Mutants in the 'Results' section) , whole cell extracts of N2a cells expressing PrP T187K and PrP T187R were digested with PK (5 痢 / ml , 37 蚓 , 30 min) and assayed by Western blotting
Similar to their chimeric counterparts and in contrast to wt PrP , mutant PrP T187K and PrP T187R showed an enhanced PK resistance leading to truncated instead of totally digested protein and , by this , confirming the results of the GFP-PrP chimeras (Fig. 6 A)
In contrast , PrP T187K and PrP T187R showed a faintly but clearly detectable overrepresentation in the pellet fraction , which indicates , in comparison to wt PrP , a higher portion of insoluble protein for these PrP mutants (Fig. 6 A)
72 h post-transfection , total protein extracts (100 痢 each at equal concentrations) of N2a cells expressing wt PrP (lanes 1 and 2) , PrP T187K (lane 3) , and PrP T187R (lane 4) were treated (+) or not treated (-) with 5 痢 / ml PK for 30 min at 37 蚓 and analyzed by immunoblotting with monoclonal anti-PrP antibody 6H4
B , whole cell extracts of N2a cells expressing wt PrP (lanes 1 and 2) , PrP T187K (lanes 3 and 4) , PrP T187R (lanes 5 and 6) , PrP W144 Stop (W144X , lanes 7 and 8) , and PrP Q159 Stop (Q159X , lanes 9 and 10) were high speed-centrifuged at 4 蚓 for 100 min to separate detergent-soluble from detergent-insoluble molecules
All experiments involving the proteasome inhibitors were also done for wt PrP , PrP T187K , and PrP T187R , but no significant increase (or decrease) after application of MG-115 or lactacystin could be detected in a Western blot or in CLSM analysis (data not shown) , indicating a non-proteasomal degradation of these full-length PrPs
Both chimeric PrP counterparts used in this study , GFP-PrP T187K and GFP-PrP T187R , showed a cellular phenotype similar to the other disease-related full-length chimeric PrP mutants , since they were properly glycosylated and transported via the secretory pathway to the cell surface
Although a strongly reduced susceptibility of surface PrPs to PIPLC has been described in some (7 , 36 ) but not all (19 ) studies for disease-related PrP mutants , we were unable to detect an impaired or delayed PIPLC releasability of GFP-PrP T187K and GFP-PrP T187R from the cell membrane in comparison to GFP-wtPrP by direct microscopic observation
However , despite the unimpaired PIPLC susceptibility of chimeric PrP T187K and PrP T187R , both PrP mutants (native as well as chimeric) showed an enhanced PK resistance and increased detergent-insolubility compared with the wt controls , which clearly indicates a higher degree of aggregation characteristically for the disease-associated PrP isoform (7 , 17 , 25 )
P101L (primers 1 and 2) , W144Stop (primers -->and 4) , Q159Stop (primers 5 and 6) , D177N (primers 7 and 8) , T187K (primers 9 and 10) , T187R (primers 11 and 12) , and E199K (primers 13 and 14) mutations in the mouse prion protein open reading frame were obtained by a PCR-based sitedirected mutagenesis method (14) with pBs-Prnp as template using the following primers: 5 -TCAGCAAACCAAAAACC-3 (primer 1) , 5 -GCTTGTTCCACTGATTA-3 (primer 2) , 5 -CCATTTTGGCAACGACTAGGAGGACCGCTACTACCG-3 (primer 3) , 5 -ATCATGGGCCTGCTCACGGCGCTCCCC-3 (primer 4) , 5 -GTACCGCTACCCTAACTAAGTGTACTACAGGCC-3 (primer 5) , 5 -ATGTTTTCACGGTAGTAGCGGTCCTCCCAGTCG-3 (primer 6) 5 -AACTGCGTCAATATCACC-3 (primer 7) , 5 -GTGCACGAAGTTGTTCTG-3 (primer 8) , 5 -CCATCAAGCAGCACAAGGTCACCACCACCACC-3 (primer 9) , 5 -TGATATTGACGCAGTCGTGCACGAAG-3 (primer 10) , 5 -GGGTCACCACCACCACCAAG-3 (primer 11) , 5 -TGTGCTGCTTGATGGTGATATTG-3 (primer 12) , 5 AAGACCGATGTGAAGATG-3 (primer 13) , 5 -GGTGAAGTTCTCCCCCT-3 (primer 14)
GFP-PrP chimera with mouse PrPa Human PrP Phenotype P101L P102L GSS W144Stopb Y145Stop GSS Q159Stop Q160Stop EODc D177N D178N FFI T187K T188K CJD T187R T188R CJDd E199K E200K CJD a Mouse PrP contains methionine at position 128 , which corresponds to codon 129 in human PrP , a polymorphic site encoding either methionine or valine
B , whole cell extracts of N2a cells expressing wt PrP (lanes 1 and 2) , PrP T187K (lanes -->and 4) , PrP T187R (lanes 5 and 6) , PrP W144Stop (W144X , lanes 7 and 8) , and PrP Q159Stop (Q159X , lanes 9 and 10) were high speed-centrifuged at 4 蚓 for 100 min to separate detergent-soluble from detergent-insoluble molecules

Reference #1 (Lorenz H et al.): T187R

P101L (primers 1 and 2) , W144 Stop (primers 3 and 4) , Q159 Stop (primers 5 and 6) , D177N (primers 7 and 8) , T187K (primers 9 and 10) , T187R (primers 11 and 12) , and E199K (primers 13 and 14) mutations in the mouse prion protein open reading frame were obtained by a PCR-based site-directed mutagenesis method (14 ) with pBs-Prnp as template using the following primers: 5'-TCAGCAAACCAAAAACC-3' (primer 1) , 5'-GCTTGTTCCACTGATTA-3' (primer 2) , 5'-CCATTTTGGCAACGACTAGGAGGACCGCTACTACCG-3' (primer 3) , 5'-ATCATGGGCCTGCTCACGGCGCTCCCC-3' (primer 4) , 5'-GTACCGCTACCCTAACTAAGTGTACTACAGGCC-3' (primer 5) , 5'-ATGTTTTCACGGTAGTAGCGGTCCTCCCAGTCG-3' (primer 6) 5'-AACTGCGTCAATATCACC-3' (primer 7) , 5'-GTGCACGAAGTTGTTCTG-3' (primer 8) , 5'-CCATCAAGCAGCACAAGGTCACCACCACCACC-3' (primer 9) , 5'-TGATATTGACGCAGTCGTGCACGAAG-3' (primer 10) , 5'-GGGTCACCACCACCACCAAG-3' (primer 11) , 5'-TGTGCTGCTTGATGGTGATATTG-3' (primer 12) , 5'-AAGACCGATGTGAAGATG-3' (primer 13) , 5'-GGTGAAGTTCTCCCCCT-3' (primer 14)
Among these PrP mutants , there were three , GFP-PrP Q159 Stop , GFP-PrP T187K , and GFP-PrP T187R (corresponding to human PrP Q160 Stop , T188K , T188R) , that have so far never been cellularly examined and which were assayed in this study most intensively
After expression in N2a cells , the chimeric PrP missense mutants , i.e. GFP-PrP P101L , GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K , were fully glycosylated and synthesized to molecular masses of about 48-58 kDa like the chimera with wt PrP (Fig. 2 A)
Lane 1 , GFP-wtPrP ; lanes 2-8 , GFP-PrP mutants with amino acid substitutions P101L (lane 2) , W144 Stop (W144X , lane 3) , Q159 Stop (Q159X , lane 4) , D177N (lane 5) , T187K (lane 6) , T187R (lane 7) , E199K (lane 8)
Lanes 1 and 2 , untreated (-) and treated (+) GFP-wtPrP ; Lanes 3-7 , treated GFP-PrP mutants with amino acid substitutions P101L (lane 3) , D177N (lane 4) , T187K (lane 5) , T187R (lane 6) , and E199K (lane 7)
Single confocal sections of cells expressing GFP-wtPrP , GFP-PrP P101L , GFP-PrP W144 Stop (W144X) , GFP-PrP Q159 Stop (Q159X , from left to right , upper panel) and GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K (from left to right , lower panel) 48 h post-transfection
The microscopic examination revealed that the chimeric mutant proteins GFP-PrP P101L , GFP-PrP D177N , GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP E199K showed no difference in localization from GFP-wtPrP (Fig. 2 C)
Cellular Trafficking of the Chimeric PrP Mutants-- The three chimeric PrP mutants GFP-PrP T187K , GFP-PrP T187R , and GFP-PrP Q159 Stop were further analyzed with respect to their cellular transport
This BFA-induced phenotype was also detected in N2a cells expressing GFP-PrP T187K and GFP-PrP T187R (Fig. 3 B) but was never observed for GFP-PrP Q159 Stop-expressing cells (Fig. 3 A)
Transfected cells showed the redistribution of GFP-PrP T187K and GFP-PrP T187R into the ER in the presence of BFA and a reorganization of discrete fluorescent signals or Golgi signals , respectively , in the absence of BFA , which clearly indicates a secretory pathway along the ER / Golgi complex for these two chimeric PrP mutants (Fig. 3 B)
The chimeric mutant PrPs T187K and T187R reveal a BFA-sensitive cellular phenotype like wt PrP , whereas chimeric PrP Q159 Stop (Q159X) localization is not influenced by BFA-incubation
B , signal (black) distributions of cells expressing (from left to right) GFP-wtPrP , GFP-PrP Q159 Stop (Q159X) , GFP-PrP T187K , GFP-PrP T187R
During PIPLC treatment for up to 40 min , the GFP-wtPrP- , GFP-PrP T187K- and GFP-PrP T187R-expressing cells showed an ongoing disappearance of the fluorescent signals on the cell surface , whereas no loss of fluorescence was observed for cells expressing GFP-PrP Q159 Stop (Fig. 4 )
Chimeric wt PrP and mutant PrP T187K and T187R (first , third , and fourth panel) could be cleaved off from the cell surface by incubation of the cells with PIPLC (1 unit / ml) for 40 min at 37 蚓
Western blotting revealed that the expression in N2a cells yielded glycosylated wt PrP , PrP T187K , and PrP T187R with molecular masses of ~27-37 kDa (Fig. 5 A)
A , N2a cells expressing wt PrP , PrP W144 Stop (W144X) , PrP Q159 Stop (Q159X) , PrP T187K , and PrP T187R (lanes 1-5) were extracted 48 h post-transfection , and equal amounts of total protein (100 痢) were subjected to Western blot analysis with anti-PrP antiserum Kan72
B , amount of secreted PrP in N2a cells expressing wt PrP , PrP W144 Stop (W144X) , PrP Q159 Stop (Q159X) , PrP T187K , and PrP T187R (lanes 1-5)
Under these conditions , secretion into the medium could easily be detected for wt PrP , PrP T187K , and PrP T187R but not for PrP W144 Stop and PrP Q159 Stop (Fig. 5 B) , supporting our data that the PrP nonsense mutants were strictly restricted to intracellular regions and , in contrast to the other examined PrPs , not trafficked along a secretory pathway
Biochemical Properties of the PrP Mutants-- To investigate whether PrP T187K and PrP T187R also exhibit an enhanced PK resistance like many other disease-associated full-length PrP mutants (7 , 8 ) or their chimeric PrP homologues , respectively (see Expression of Chimeric Prp Mutants in the 'Results' section) , whole cell extracts of N2a cells expressing PrP T187K and PrP T187R were digested with PK (5 痢 / ml , 37 蚓 , 30 min) and assayed by Western blotting
Similar to their chimeric counterparts and in contrast to wt PrP , mutant PrP T187K and PrP T187R showed an enhanced PK resistance leading to truncated instead of totally digested protein and , by this , confirming the results of the GFP-PrP chimeras (Fig. 6 A)
In contrast , PrP T187K and PrP T187R showed a faintly but clearly detectable overrepresentation in the pellet fraction , which indicates , in comparison to wt PrP , a higher portion of insoluble protein for these PrP mutants (Fig. 6 A)
72 h post-transfection , total protein extracts (100 痢 each at equal concentrations) of N2a cells expressing wt PrP (lanes 1 and 2) , PrP T187K (lane 3) , and PrP T187R (lane 4) were treated (+) or not treated (-) with 5 痢 / ml PK for 30 min at 37 蚓 and analyzed by immunoblotting with monoclonal anti-PrP antibody 6H4
B , whole cell extracts of N2a cells expressing wt PrP (lanes 1 and 2) , PrP T187K (lanes 3 and 4) , PrP T187R (lanes 5 and 6) , PrP W144 Stop (W144X , lanes 7 and 8) , and PrP Q159 Stop (Q159X , lanes 9 and 10) were high speed-centrifuged at 4 蚓 for 100 min to separate detergent-soluble from detergent-insoluble molecules
All experiments involving the proteasome inhibitors were also done for wt PrP , PrP T187K , and PrP T187R , but no significant increase (or decrease) after application of MG-115 or lactacystin could be detected in a Western blot or in CLSM analysis (data not shown) , indicating a non-proteasomal degradation of these full-length PrPs
Both chimeric PrP counterparts used in this study , GFP-PrP T187K and GFP-PrP T187R , showed a cellular phenotype similar to the other disease-related full-length chimeric PrP mutants , since they were properly glycosylated and transported via the secretory pathway to the cell surface
Although a strongly reduced susceptibility of surface PrPs to PIPLC has been described in some (7 , 36 ) but not all (19 ) studies for disease-related PrP mutants , we were unable to detect an impaired or delayed PIPLC releasability of GFP-PrP T187K and GFP-PrP T187R from the cell membrane in comparison to GFP-wtPrP by direct microscopic observation
However , despite the unimpaired PIPLC susceptibility of chimeric PrP T187K and PrP T187R , both PrP mutants (native as well as chimeric) showed an enhanced PK resistance and increased detergent-insolubility compared with the wt controls , which clearly indicates a higher degree of aggregation characteristically for the disease-associated PrP isoform (7 , 17 , 25 )
P101L (primers 1 and 2) , W144Stop (primers -->and 4) , Q159Stop (primers 5 and 6) , D177N (primers 7 and 8) , T187K (primers 9 and 10) , T187R (primers 11 and 12) , and E199K (primers 13 and 14) mutations in the mouse prion protein open reading frame were obtained by a PCR-based sitedirected mutagenesis method (14) with pBs-Prnp as template using the following primers: 5 -TCAGCAAACCAAAAACC-3 (primer 1) , 5 -GCTTGTTCCACTGATTA-3 (primer 2) , 5 -CCATTTTGGCAACGACTAGGAGGACCGCTACTACCG-3 (primer 3) , 5 -ATCATGGGCCTGCTCACGGCGCTCCCC-3 (primer 4) , 5 -GTACCGCTACCCTAACTAAGTGTACTACAGGCC-3 (primer 5) , 5 -ATGTTTTCACGGTAGTAGCGGTCCTCCCAGTCG-3 (primer 6) 5 -AACTGCGTCAATATCACC-3 (primer 7) , 5 -GTGCACGAAGTTGTTCTG-3 (primer 8) , 5 -CCATCAAGCAGCACAAGGTCACCACCACCACC-3 (primer 9) , 5 -TGATATTGACGCAGTCGTGCACGAAG-3 (primer 10) , 5 -GGGTCACCACCACCACCAAG-3 (primer 11) , 5 -TGTGCTGCTTGATGGTGATATTG-3 (primer 12) , 5 AAGACCGATGTGAAGATG-3 (primer 13) , 5 -GGTGAAGTTCTCCCCCT-3 (primer 14)
GFP-PrP chimera with mouse PrPa Human PrP Phenotype P101L P102L GSS W144Stopb Y145Stop GSS Q159Stop Q160Stop EODc D177N D178N FFI T187K T188K CJD T187R T188R CJDd E199K E200K CJD a Mouse PrP contains methionine at position 128 , which corresponds to codon 129 in human PrP , a polymorphic site encoding either methionine or valine
B , whole cell extracts of N2a cells expressing wt PrP (lanes 1 and 2) , PrP T187K (lanes -->and 4) , PrP T187R (lanes 5 and 6) , PrP W144Stop (W144X , lanes 7 and 8) , and PrP Q159Stop (Q159X , lanes 9 and 10) were high speed-centrifuged at 4 蚓 for 100 min to separate detergent-soluble from detergent-insoluble molecules

F.Horn (priondbcmbi.ru.nl), 22-Aug-2005