ABCMdb

ABCC4 p.Thr796Met

None has been submitted yet.

Publications

PMID: 23766510 [PubMed] Cheepala SB et al: "Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression."

No. Sentence Comment

7 We demonstrate that zebrafish Abcc4 T804M or human ABCC4 T796M exhibit substantially reduced expression, coupled with impaired plasma membrane localization. Login to comment
37 Our studies demonstrate that the analogous mutation in human ABCC4 (T796M) also reduces protein expression. Login to comment
47 In Vitro Translation-In vitro translation reactions were performed with the following expression plasmids: pcDNA3.1 ZF Abcc4, Abcc4 T804M, ABCC4, ABCC4 T796M, and pLUC (luciferase vector) using the transcription-and translation-coupled (TNT) kit (Promega Inc., Madison, WI) in the presence of [35 S]Met. Login to comment
51 Cell Culture-NIH3T3, HEK293, or the cells transiently expressing one of the following proteins, ZF Abcc4, ZF Abcc4 T804M, ABCC4, and ABCC4 T796M, were maintained in Dulbecco`s DMEM containing 4500 mg/liter glucose, 10% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, and 100 òe;g/ml streptomycin in a humidified 5% CO2 incubator at 37 &#b0;C. Login to comment
52 Where specified, NIH3T3 cells transiently expressing either pACGFP ABCC4 or T796M were incubated with 50 òe;g/ml cycloheximide for different time points. Login to comment
67 Confocal Microscopy-NIH3T3 or HEK293 cells were seeded on coverslips and chamber slides 24 h before transfection with the plasmid DNAs (250 ng) encoding pAcGFP ZF Abcc4, ZF Abcc4 T804M, ABCC4, ABCC4 T796M, and pCAL plasmid encoding RFP-tagged endoplasmic reticulum resident protein calreticulin encoding plasmid (pCAL (Origene) by using Lipofectamine LTX Plus reagent (Invitrogen))according to the manufacturer`s protocols. Login to comment
72 Briefly, NIH3T3 cells transiently expressing either pAcGFP human ABCC4 or T796M were rinsed with ice-cold PBS and incubated with 10 mM EZ-Link sulfo-NHS-SS-biotin solution in PBS (pH 8.0) (Pierce) for 30 min at room temperature. Login to comment
103 H, loss of intermolecular interactions of T796M human ABCC4. Login to comment
122 Because the Thr at position 804 in ZF Abcc4 is conserved among Abcc4 subfamily members, we developed expression vectors encoding full-length human WT AcGFP-ABCC4 and produced an analogous mutation to T804M, T796M in PAcGFP ABCC4. Login to comment
124 ABCC4 T796M exhibited a strong reduction in the core-glycosylated mature band c (Fig. 2F), which was confirmed by treating WT ABCC4-expressing lysates with the N-glycanase, PNGase F (Fig. 2G). Login to comment
125 Notably, WT human ABCC4 and T796M produced almost identical amounts of protein (Fig. 2H) when in vitro translated, indicating that, like ZF T804M, the T796M substitution in human ABCC4 has no effect on mRNA translation. To determine T796M ABCC4 subcellular localization, live cell confocal microscopy was performed. Login to comment
126 Cells were transiently transfected with either ABCC4 WT or T796M expression plasmids along with an expression plasmid for calreticulin, an ER resident protein. Login to comment
129 In contrast, a majority (&#2b0e;95%) of ABCC4 T796M localized with the Golgi/ER marker, calreticulin, or more diffusely in the cytoplasm (Fig. 2, I and J). Login to comment
130 We next determined if the T796M substitution in CL3 altered the stability of human ABCC4. Login to comment
131 NIH3T3 cells were transiently transfected with either WT ABCC4 or ABCC4 harboring T796M substitution. Login to comment
134 In contrast, ABCC4 T796M does not have a readily detectable mature ABCC4 (band c); however, the turnover of the immature ABCC4 (band b) for WT and T796M are almost identical, suggesting the immature form of ABCC4 and T796M ABCC4 has the same degradation rate. Login to comment
135 Cytoplasmic Loop 3 Helicity Is Altered and Associated with Temperature-sensitive Rescue-We hypothesized that the T796M mutation in the human ABCC4 affects the helical conformation of the CL3 region. Login to comment
137 The helix containing T796M substitution was iteratively interrogated 16 times, and the structures were determined at temperatures ranging from 270 to 602 K. Login to comment
148 F, immunoblots of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 WT and ABCC4 T796M mutant. Login to comment
152 J, quantification of HEK293 transfected cells with GFP-tagged human ABCC4 WT, ABCC T796M mutant, and RFP-tagged ER marker (calreticulin). Login to comment
154 Signals from the three channels were acquired independently, and the merged images are presented. Co-localization of ABCC4 WT and PM is indicated by a reddish blue, and co-localization of GFP ABCC4 T796M and RFP-ER is indicated by a yellow color. Login to comment
155 K, NIH3T3 cells were transfected with ABCC4 WT, T796M, and cycloheximide (final concentration, 50 òe;g/ml) was added 24 h post-transfection. Login to comment
159 T796M mutant, indicating that the stability of Met-796 containing helix is worse than the wild type. Login to comment
161 Based on the MD analysis, we hypothesized that expression of the ABCC4 T796M might be restored by reducing temperature to 28 &#b0;C. Login to comment
162 We next investigated if subphysiological temperatures rescued expression and plasma membrane localization of ABCC4 T796M. Login to comment
163 NIH3T3 cells were transiently transfected with either ABCC4 or T796M expression vectors; 24 h post-transfection, one set of cells was transferred to 28 &#b0;C incubation, and the other remained at 37 &#b0;C. Login to comment
166 For the total protein, T796M is reduced by 50% compared with ABCC4 at 37 &#b0;C, with the unexpected increase in the amount of the mature form ABCC4 T796M likely due to the additional 24-h incubation. Login to comment
167 Interestingly, the immature band b in T796M was dramatically reduced by this additional incubation period. Login to comment
168 However, at 28 &#b0;C, T796M expression and surface expression are strongly increased (Fig. 3, B and C). Login to comment
169 This result indicates that subphysiological temperatures can rescue T796M expression and restore plasma membrane localization. Login to comment
171 Human ABCC4 T796M peptide forms an unstable helix. Login to comment
172 A, percentage of ॷ-helix is plotted versus temperature (K) from the data derived from in silico molecular dynamics analysis of WT human ABCC4 and T796M mutant peptide. B and C, immunoblot of surface-biotinylated streptavidin-bound lysates (100 òe;g of protein/lane) prepared from NIH3T3 cells exposed to 37 or 28 &#b0;C for 24 h after 24 h of transfection with WT (human ABCC4) and T796M and its quantification. Login to comment
177 Next, we directly determined the effect of T796M mutation on the helix forming capabilities of a fragment of CL3(783-808), using circular dichroism spectrophotometry on CL3 peptides from human WT ABCC4 or T796M CL3 using approaches we previously reported (42). Login to comment
180 In contrast, the ABCC4 peptide harboring the ABCC4 T796M mutation required substantially higher concentrations of TFE to elicit comparable changes in helicity. Login to comment
181 These results are compatible with our molecular dynamics simulations (Fig. 3A) and provide additional confirmation that the T796M mutation alters the helicity of this region of CL3 in ABCC4. Login to comment
183 We observed that the helical content of T804M was consistently lower than the ZF Abcc4 at temperatures greater than 301 K (Fig. 4A), results that are similar to T796M (Fig. 3A). Login to comment
186 The Abcc4 and Abcc4 T804M proteins each increased expression at 28 &#b0;C; however, for Abcc4 T804M the mature band c is difficult to resolve (Fig. 4B), and instead we observed a strong increase in protein, a result that is qualitatively similar to human T796M. Login to comment
202 ABCC4 S794L and H796Y as well as ZF Abcc4 A802L and H806Y substitutions affected both protein expression and highly reduced the levels of the mature band c (Fig. 5, C and E). Login to comment
203 Notably, the expression pattern of these mutants with reduced mature glycosylated band c is similar to ABCC4 T796M (Fig. 5E). Login to comment
210 The R815A mutation resulted in reduced ABCC4 expression and loss of mature glycosylated band c (Fig. 6, B and C). Login to comment
211 The reduction in mature ABCC4 is consistent with the prediction that CL3 Arg-815 interacts with the NBD to ensure proper folding of the protein To test if reduced temperature enhanced expression of R815A, NIH3T3 cells were transiently transfected with ABCC4, T796M-ABCC4, or R815A-ABCC4 and then incubated at 28 &#b0;C. Login to comment
215 However, in ZF and human ABCC4 (T796M, is the analogous position), protein FIGURE 4. Login to comment
240 The reduced amounts of mutant ABCC4 protein (T804M in zebrafish and T796M in humans) are not related to an impaired translation of the ABCC4 mRNA, which is almost identical among WT and T796M mRNA despite an apparent alteration in the energetics of mRNA folding. Login to comment
241 It is likely that disrupted processing in the ER of ABCC4 is disrupted by the T796M substitution. Login to comment
244 Like the T796M for human and the orthologous T804M in zebrafish, these addi- FIGURE 5. Login to comment
249 E, immunoblot of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 and ABCC4 mutants (T796M, S794L, and H798Y), and these blots were probed with anti-GFP antibody. Login to comment
257 The increased formation of the mature c-form of ABCC4 R815A at the subphysiological temperature of 28 &#b0;C is consistent with the proposal that CL3/NBD1 interactions are necessary for proper folding of ABCC4. Login to comment
258 In further support of ABCC4 CL3 mutants being misfolded, preliminary studies show that that two small molecules that reduce the activity of the ER unfolded protein response (phenylbutyrate (29) and glycerol (36)) selectively rescue expression of the ABCC4 CL3 T796M (data not shown). Login to comment
260 In cells harboring ABCC4, a T796M substitution, reduced amounts of the mature glycosylated form are observed. Login to comment
261 These findings, and the internal mislocalization of T796M, suggest that either ER glycosylation of ABCC4 is impaired or the protein is mostly degraded before maturation is complete. Login to comment
262 We show that T796M has reduced mature ABCC4 (c-form) coupled with decreased abundance at the plasma membrane. Login to comment
263 We favor the idea that the T796M substitution does not generally produce defective glycosylation of ABCC4 because plasma membrane localization and ABCC4 glycosyl maturation are mostly restored at reduced temperatures. Login to comment
264 However, we cannot rule out the possibility that the increased expression of T796M at subphysiological temperatures might be due, in part, to reduced protease activity (it seems unlikely that reduced protease activity modifies glycosylation). Login to comment
270 These findings buttress our molecular dynamics simulations showing that the region containing T796M has less thermal stability, a finding consistent with this region and with CL3`s reduced ability to form an ॷ-helix. Login to comment
35 Our studies demonstrate that the analogous mutation in human ABCC4 (T796M) also reduces protein expression. Login to comment
45 In Vitro Translation-In vitro translation reactions were performed with the following expression plasmids: pcDNA3.1 ZF Abcc4, Abcc4 T804M, ABCC4, ABCC4 T796M, and pLUC (luciferase vector) using the transcription- and translation-coupled (TNT) kit (Promega Inc., Madison, WI) in the presence of [35 S]Met. Login to comment
49 Cell Culture-NIH3T3, HEK293, or the cells transiently expressing one of the following proteins, ZF Abcc4, ZF Abcc4 T804M, ABCC4, and ABCC4 T796M, were maintained in Dulbecco`s DMEM containing 4500 mg/liter glucose, 10% FBS (HyClone, Logan, UT), 2 mM L-glutamine, 100 units/ml penicillin, and 100 òe;g/ml streptomycin in a humidified 5% CO2 incubator at 37 &#b0;C. Login to comment
50 Where specified, NIH3T3 cells transiently expressing either pACGFP ABCC4 or T796M were incubated with 50 òe;g/ml cycloheximide for different time points. Login to comment
65 Confocal Microscopy-NIH3T3 or HEK293 cells were seeded on coverslips and chamber slides 24 h before transfection with the plasmid DNAs (250 ng) encoding pAcGFP ZF Abcc4, ZF Abcc4 T804M, ABCC4, ABCC4 T796M, and pCAL plasmid encoding RFP-tagged endoplasmic reticulum resident protein calreticulin encoding plasmid (pCAL (Origene) by using Lipofectamine LTX Plus reagent (Invitrogen))according to the manufacturer`s protocols. Login to comment
71 Briefly, NIH3T3 cells transiently expressing either pAcGFP human ABCC4 or T796M were rinsed with ice-cold PBS and incubated with 10 mM EZ-Link sulfo-NHS-SS-biotin solution in PBS (pH 8.0) (Pierce) for 30 min at room temperature. Login to comment
102 H, loss of intermolecular interactions of T796M human ABCC4. Login to comment
121 Because the Thr at position 804 in ZF Abcc4 is conserved among Abcc4 subfamily members, we developed expression vectors encoding full-length human WT AcGFP-ABCC4 and produced an analogous mutation to T804M, T796M in PAcGFP ABCC4. Login to comment
123 ABCC4 T796M exhibited a strong reduction in the core-glycosylated mature band c (Fig. 2F), which was confirmed by treating WT ABCC4-expressing lysates with the N-glycanase, PNGase F (Fig. 2G). Login to comment
128 In contrast, a majority (b0e;95%) of ABCC4 T796M localized with the Golgi/ER marker, calreticulin, or more diffusely in the cytoplasm (Fig. 2, I and J). Login to comment
133 In contrast, ABCC4 T796M does not have a readily detectable mature ABCC4 (band c); however, the turnover of the immature ABCC4 (band b) for WT and T796M are almost identical, suggesting the immature form of ABCC4 and T796M ABCC4 has the same degradation rate. Login to comment
136 The helix containing T796M substitution was iteratively interrogated 16 times, and the structures were determined at temperatures ranging from 270 to 602 K. Login to comment
147 F, immunoblots of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 WT and ABCC4 T796M mutant. Login to comment
151 J, quantification of HEK293 transfected cells with GFP-tagged human ABCC4 WT, ABCC T796M mutant, and RFP-tagged ER marker (calreticulin). Login to comment
153 Signals from the three channels were acquired independently, and the merged images are presented. Co-localization of ABCC4 WT and PM is indicated by a reddish blue, and co-localization of GFP ABCC4 T796M and RFP-ER is indicated by a yellow color. Login to comment
158 T796M mutant, indicating that the stability of Met-796 containing helix is worse than the wild type. Login to comment
160 Based on the MD analysis, we hypothesized that expression of the ABCC4 T796M might be restored by reducing temperature to 28 &#b0;C. Login to comment
165 For the total protein, T796M is reduced by 50% compared with ABCC4 at 37 &#b0;C, with the unexpected increase in the amount of the mature form ABCC4 T796M likely due to the additional 24-h incubation. Login to comment
170 Human ABCC4 T796M peptide forms an unstable helix. Login to comment
176 Next, we directly determined the effect of T796M mutation on the helix forming capabilities of a fragment of CL3(783-808), using circular dichroism spectrophotometry on CL3 peptides from human WT ABCC4 or T796M CL3 using approaches we previously reported (42). Login to comment
179 In contrast, the ABCC4 peptide harboring the ABCC4 T796M mutation required substantially higher concentrations of TFE to elicit comparable changes in helicity. Login to comment
182 We observed that the helical content of T804M was consistently lower than the ZF Abcc4 at temperatures greater than 301 K (Fig. 4A), results that are similar to T796M (Fig. 3A). Login to comment
185 The Abcc4 and Abcc4 T804M proteins each increased expression at 28 &#b0;C; however, for Abcc4 T804M the mature band c is difficult to resolve (Fig. 4B), and instead we observed a strong increase in protein, a result that is qualitatively similar to human T796M. Login to comment
214 However, in ZF and human ABCC4 (T796M, is the analogous position), protein FIGURE 4. Login to comment
239 The reduced amounts of mutant ABCC4 protein (T804M in zebrafish and T796M in humans) are not related to an impaired translation of the ABCC4 mRNA, which is almost identical among WT and T796M mRNA despite an apparent alteration in the energetics of mRNA folding. Login to comment
243 Like the T796M for human and the orthologous T804M in zebrafish, these addi- FIGURE 5. Login to comment
248 E, immunoblot of whole cell lysates (100 òe;g of protein per lane) prepared from NIH3T3 cells transfected with human ABCC4 and ABCC4 mutants (T796M, S794L, and H798Y), and these blots were probed with anti-GFP antibody. Login to comment
259 In cells harboring ABCC4, a T796M substitution, reduced amounts of the mature glycosylated form are observed. Login to comment
269 These findings buttress our molecular dynamics simulations showing that the region containing T796M has less thermal stability, a finding consistent with this region and with CL3`s reduced ability to form an ॷ-helix. Login to comment