Prestin is an essential component of the molecular motor of cochlear outer hair cells that contri... more Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane. We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature. Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC. Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.
We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein... more We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.
Biochemical and Biophysical Research Communications, 1988
We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the ... more We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the androgen receptor. The protein is detected in the soluble fraction by two-dimensional polyacrylamide gel electrophoresis as two spots with isoelectric points of 6.7 and 6.5 respectively. Photoaffinity labelling of GSF with 8 nM or 50 nM [3H]-methyltrienolone selectively labels the two protein spots but with an apparently lower affinity than is known for the androgen receptor. The two protein spots stem from one protein as judged from peptide patterns of partial proteolytic digests and the equal labelling of both spots with methyltrienolone. Cells from subjects with mutant androgen receptors generally lack the 56 kD protein and labelling with methyltrienolone fails, but the protein is not the androgen receptor itself. We propose the hypothesis that the 56 kD protein is synthesized from the same gene as the androgen receptor and that androgen may not be its natural ligand.
Journal of Steroid Biochemistry and Molecular Biology, 1996
COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologu... more COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologues have been cloned in several species, from Drosophila to man. The vertebrate COUP-TFs can be classified into four subgroups according to sequence homology in their ligand-binding domain. COUP-TFs bind to AGGTCA direct repeats or palindromes with various spacings. These include the response elements of several other members of
The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives co... more The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives cochlear amplification and produces exquisitely sharp frequency tuning. Tecta(C1509G) transgenic mice have hearing loss, and surprisingly have increased OHC prestin levels. We hypothesized, therefore, that prestin up-regulation may represent a generalized response to compensate for a state of hearing loss. In the present study, we sought to determine the effects of noise-induced hearing loss on prestin expression. After noise exposure, we performed cytocochleograms and observed OHC loss only in the basal region of the cochlea. Next, we patch clamped OHCs from the apical turn (9-12 kHz region), where no OHCs were lost, in noise-exposed and age-matched control mice. The non-linear capacitance was significantly higher in noise-exposed mice, consistent with higher functional prestin levels. We then measured prestin protein and mRNA levels in whole-cochlea specimens. Both Western blot and qPCR ...
Prestin is an essential component of the molecular motor of cochlear outer hair cells that contri... more Prestin is an essential component of the molecular motor of cochlear outer hair cells that contribute to frequency selectivity and sensitivity of mammalian hearing. A model system to study prestin employs its transfection into cultured HEK 293 cells. Our goal was to characterize prestin's trafficking pathway and localization in the plasma membrane. We used immuno-colocalization of prestin with intracellular and plasma membrane markers and sucrose density fractionation to analyze prestin in membrane compartments. Voltage clamping was used to measure nonlinear capacitance (NLC), prestin's electrical signature. Prestin targets to the membrane by 24 hours post-transfection when NLC is measurable. Prestin then concentrates into membrane foci that colocalize and fractionate with membrane microdomains. Depleting membrane cholesterol content altered prestin localization and NLC. Prestin activity in HEK 293 cells results from expression in the plasma membrane and altering membrane lipid content affects prestin localization and activity.
We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein... more We have recently described in genital skin fibroblasts (GSF) a relatively abundant 56 kDa protein with androgen-binding activity. This protein is missing in GSF of most patients with complete androgen insensitivity syndrome (CAI). The protein has many characteristics compatible with the androgen receptor; it has in fact been tentatively considered as a precursor or degradation form of the prototypic (approximately 100 kDa) human androgen receptor. We have prepared an antiserum to this protein, which allowed us to detect it as a direct product by in vitro translation of mRNA from GSF. It is thus very unlikely to be a degradation product of a larger precursor. Furthermore, covalent photolytic labeling of this protein with the androgen analogue [3H]mibolerone revealed a much lower affinity for this protein than is known for the androgen receptor. Finally, the GSF of two exceptional patients with complete androgen insensitivity syndrome due to negligible androgen receptor-binding activity express this protein normally, as determined on two-dimensional gels by Western blot analysis with the antiserum and by photolytic covalent labeling with androgen analogues. These data indicate that the protein is not a precursor or a degradation product of the receptor; nor is it androgen-induced. They are more compatible with the idea that the protein is another member of the steroid/thyroid/retinoic acid receptor supergene family, perhaps as an unorthodox product of the human androgen receptor gene.
Biochemical and Biophysical Research Communications, 1988
We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the ... more We have found in genital skin fibroblasts an abundant 56 kD protein which appears related to the androgen receptor. The protein is detected in the soluble fraction by two-dimensional polyacrylamide gel electrophoresis as two spots with isoelectric points of 6.7 and 6.5 respectively. Photoaffinity labelling of GSF with 8 nM or 50 nM [3H]-methyltrienolone selectively labels the two protein spots but with an apparently lower affinity than is known for the androgen receptor. The two protein spots stem from one protein as judged from peptide patterns of partial proteolytic digests and the equal labelling of both spots with methyltrienolone. Cells from subjects with mutant androgen receptors generally lack the 56 kD protein and labelling with methyltrienolone fails, but the protein is not the androgen receptor itself. We propose the hypothesis that the 56 kD protein is synthesized from the same gene as the androgen receptor and that androgen may not be its natural ligand.
Journal of Steroid Biochemistry and Molecular Biology, 1996
COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologu... more COUP-TFs are orphan members of the steroid/thyroid hormone receptor superfamily. COUP-TF homologues have been cloned in several species, from Drosophila to man. The vertebrate COUP-TFs can be classified into four subgroups according to sequence homology in their ligand-binding domain. COUP-TFs bind to AGGTCA direct repeats or palindromes with various spacings. These include the response elements of several other members of
The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives co... more The outer hair cell (OHC) motor protein prestin is necessary for electromotility, which drives cochlear amplification and produces exquisitely sharp frequency tuning. Tecta(C1509G) transgenic mice have hearing loss, and surprisingly have increased OHC prestin levels. We hypothesized, therefore, that prestin up-regulation may represent a generalized response to compensate for a state of hearing loss. In the present study, we sought to determine the effects of noise-induced hearing loss on prestin expression. After noise exposure, we performed cytocochleograms and observed OHC loss only in the basal region of the cochlea. Next, we patch clamped OHCs from the apical turn (9-12 kHz region), where no OHCs were lost, in noise-exposed and age-matched control mice. The non-linear capacitance was significantly higher in noise-exposed mice, consistent with higher functional prestin levels. We then measured prestin protein and mRNA levels in whole-cochlea specimens. Both Western blot and qPCR ...
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Papers by Fred Pereira