Phenotypic differences in PFIC2 and BRIC2 correlate with protein stability of mutant Bsep and impaired taurocholate secretion in MDCK II cells

Progressive familial cholestasis (PFIC) 2 and benign recurrent intrahepatic cholestasis (BRIC) 2 are caused by mutations in the bile salt export pump (BSEP, ABCB11) gene; however, their prognosis differs. PFIC2 progresses to cirrhosis and requires liver transplantation, whereas BRIC2 is clinically benign. To identify the molecular mechanism(s) responsible for the phenotypic differences, eight PFIC2 and two BRIC2 mutations were introduced in rat Bsep, which was transfected in MDCK II cells. Taurocholate transport activity, protein expression, and subcellular distribution of these mutant proteins were studied in a polarized MDCK II monolayer. The taurocholate transport activity was approximately half of the wild-type (WT) in BRIC2 mutants (A570T and R1050C), was substantially less in two PFIC2 mutants (D482G and E297G), and was almost abolished in six other PFIC2 mutants (K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X). Bsep protein expression levels correlated closely with transport activity, except for R1057X. The half-life of the D482G mutant was shorter than that of the WT (1.35 h vs. 3.49 h in the mature form). BRIC2 mutants and three PFIC mutants (D482G, E297G, and R1057X) were predominantly distributed in the apical membrane. The other PFIC2 mutants remained intracellular. The R1057X mutant protein was stably expressed and trafficked to the apical membrane, suggesting that the COOH-terminal tail is required for transport activity but not for correct targeting. In conclusion, taurocholate transport function was impaired in proportion to rapid degradation of Bsep protein in the mutants, which were aligned in the following order: A570T and R1050C > D482G > E297G > K461E, G982R, R1153C, R1268Q, 3767–3768insC, and R1057X. These results may explain the phenotypic difference between BRIC2 and PFIC2.

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Two N-linked glycans are required to maintain the transport activity of the bile salt export pump (ABCB11) in MDCK II cells

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00415.2006 ◽

2007 ◽

Vol 292 (3) ◽

pp. G818-G828 ◽

Cited By ~ 35

Author(s):

Kaori Mochizuki ◽

Tatehiro Kagawa ◽

Asano Numari ◽

Matthew J. Harris ◽

Johbu Itoh ◽

...

Keyword(s):

Protein Stability ◽

Bile Salt ◽

Intracellular Trafficking ◽

Double Mutant ◽

Yellow Fluorescent Protein ◽

Bile Salt Export Pump ◽

Transport Activity ◽

Wild Type ◽

Mutant Proteins ◽

Quadruple Mutant

The aim of this study was to determine the role of N-linked glycosylation in protein stability, intracellular trafficking, and bile acid transport activity of the bile salt export pump [Bsep (ATP-binding cassette B11)]. Rat Bsep was fused with yellow fluorescent protein, and the following mutants, in which Asn residues of putative glycosylation sites (Asn109, Asn116, Asn122, and Asn125) were sequentially replaced with Gln, were constructed by site-directed mutagenesis: single N109Q, double N109Q + N116Q, triple N109Q + N116Q + N122Q, and quadruple N109Q + N116Q + N122Q + N125Q. Immunoblot and glycosidase cleavage analysis demonstrated that each site was glycosylated. Removal of glycans decreased taurocholate transport activity as determined in polarized MDCK II cells. This decrease resulted from rapid decay of the mutant Bsep protein; biochemical half-lives were 3.76, 3.65, 3.24, 1.35, and 0.52 h in wild-type, single-mutant, double-mutant, triple-mutant, and quadruple-mutant cells, respectively. Wild-type and single- and double-mutant proteins were distributed exclusively along the apical membranes, whereas triple- and quadruple-mutant proteins remained intracellular. MG-132 but not bafilomycin A1 extended the half-life, suggesting a role for the proteasome in Bsep degradation. To determine whether a specific glycosylation site or the number of glycans was critical for protein stability, we studied the protein expression of combinations of N-glycan-deficient mutants and observed that Bsep with one glycan was considerably unstable compared with Bsep harboring two or more glycans. In conclusion, at least two N-linked glycans are required for Bsep protein stability, intracellular trafficking, and function in the apical membrane.

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Proteasome Inhibitors Restore to Normal the Decreased Levels of Protein Expression and Nucleolar Localization of Various Mutant Ribosomal S19 Proteins Identified in DBA Patients.

Blood ◽

10.1182/blood.v106.11.185.185 ◽

2005 ◽

Vol 106 (11) ◽

pp. 185-185 ◽

Cited By ~ 3

Author(s):

Aurore Cretien ◽

Alexis Proust ◽

Hanna Gazda ◽

Jorg Meerpohl ◽

Charlotte Marie Niemeyer ◽

...

Keyword(s):

Subcellular Localization ◽

Protein Expression ◽

Proteasome Inhibitors ◽

Nucleolar Localization ◽

Wild Type ◽

Mutant Proteins ◽

Expression Levels ◽

Type Protein ◽

Wild Type Protein ◽

Proteosomal Degradation

Abstract Mutations in ribosomal protein S19 (RPS19) gene have been found in 25% of patients affected with Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia. We have previously shown that several RPS19 mutant proteins (V15F, InsAG36, W33stop, Y48stop, R56stop, M75stop, R94stop, 274del31, InsG238, G127Q and L131P) exhibit decreased levels of protein expression and do not localize to the nucleolus like the wild type protein in transfected Cos-7 cells. In contrast, other mutants (W52C, T55M, R56Q, R62W, 24Del18, G120S) exhibit normal levels of protein expression and normal nucleolar localization. We hypothesized that decreased levels of expression of mutant proteins such as V15F, G127Q, and L131P may be due to proteosomal degradation. In order to validate our hypothesis, we analyzed the effects of two proteasome inhibitors (MG132 and lactacystin) on mutant RPS19 protein expression levels and their subcellular localization. Following treatment with proteosome inhibitors, the mutant proteins with missense mutations (V15F, G127Q and L131P) were expressed at levels similar to that of wild type protein and localized in the nucleolus. Similarly, proteasome inhibitors also restored the expression levels and normal subcellular localization to RPS19 with non-sense mutations (InsG238, R94stop, and 274del31) that resulted in the translation of RPS19 protein with at least 80 aminoacids. In marked contrast, proteosome inhibitors failed to restore the expression levels of RPS19 with the non-sense mutants that led to synthesis of shortened proteins (InsAG36, W33stop, Y48stop, R56stop, M75stop). Even in the presence of proteosome inhibitors we noted a dramatic decrease in the levels of expression of these mutant proteins and proteins expressed were localized in the cytoplasm. Our findings imply an important role for proteosomal degradation pathway in regulating the expression levels of RPS19. They further suggest that proteasome inhibitors could be considered as a potential treatment for some steroid resistant DBA affected individuals with specific RPS19 mutations.

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Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen

Neurology International ◽

10.3390/neurolint14010002 ◽

2021 ◽

Vol 14 (1) ◽

pp. 11-33

Author(s):

Sui Sawaguchi ◽

Kenji Tago ◽

Hiroaki Oizumi ◽

Katsuya Ohbuchi ◽

Masahiro Yamamoto ◽

...

Keyword(s):

Protein Expression ◽

Rna Polymerase ◽

Rna Polymerase Iii ◽

Mtor Signaling ◽

Marker Protein ◽

Wild Type ◽

Mutant Proteins ◽

Oligodendroglial Cell ◽

Hypomyelinating Leukodystrophy ◽

In Cells

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD−102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.

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Functional and mechanistic roles of the human proton-coupled folate transporter transmembrane domain 6–7 linker

Biochemical Journal ◽

10.1042/bcj20160399 ◽

2016 ◽

Vol 473 (20) ◽

pp. 3545-3562 ◽

Cited By ~ 2

Author(s):

Mike R. Wilson ◽

Zhanjun Hou ◽

Lucas J. Wilson ◽

Jun Ye ◽

Larry H. Matherly

Keyword(s):

Secondary Structure ◽

Targeted Delivery ◽

Transmembrane Domain ◽

Surface Expression ◽

Chimeric Proteins ◽

Transport Activity ◽

Wild Type ◽

Intracellular Loop ◽

Mutant Proteins ◽

Sequence Elements

The proton-coupled folate transporter (PCFT; SLC46A1) is a folate–proton symporter expressed in solid tumors and is used for tumor-targeted delivery of cytotoxic antifolates. Topology modeling suggests that the PCFT secondary structure includes 12 transmembrane domains (TMDs) with TMDs 6 and 7 linked by an intracellular loop (positions 236–265) including His247, implicated as functionally important. Single-cysteine (Cys) mutants were inserted from positions 241 to 251 in Cys-less PCFT and mutant proteins were expressed in PCFT-null (R1-11) HeLa cells; none were reactive with 2-aminoethyl methanethiosulfonate biotin, suggesting that the TMD6–7 loop is intracellular. Twenty-nine single alanine mutants spanning the entire TMD6–7 loop were expressed in R1-11 cells; activity was generally preserved, with the exception of the 247, 250, and 251 mutants, partly due to decreased surface expression. Coexpression of PCFT TMD1–6 and TMD7–12 half-molecules in R1-11 cells partially restored transport activity, although removal of residues 252–265 from TMD7–12 abolished transport. Chimeric proteins, including a nonhomologous sequence from a thiamine transporter (ThTr1) inserted into the PCFT TMD6–7 loop (positions 236–250 or 251–265), were active, although replacement of the entire loop with the ThTr1 sequence resulted in substantial loss of activity. Amino acid replacements (Ala, Arg, His, Gln, and Glu) or deletions at position 247 in wild-type and PCFT–ThTr1 chimeras resulted in differential effects on transport. Collectively, our findings suggest that the PCFT TMD6–7 connecting loop confers protein stability and may serve a unique functional role that depends on secondary structure rather than particular sequence elements.

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AutoNeuriteJ: An ImageJ plugin for measurement and classification of neuritic extensions

10.1101/2020.05.29.123000 ◽

2020 ◽

Author(s):

Benoit Boulan ◽

Anne Beghin ◽

Charlotte Ravanello ◽

Jean-Christophe Deloulme ◽

Sylvie Gory-Fauré ◽

...

Keyword(s):

Neuronal Cultures ◽

Neuronal Growth ◽

Wild Type ◽

Phenotypic Difference ◽

Phenotypic Differences ◽

Large Numbers ◽

Neuritic Growth ◽

Imagej Plugin

AbstractMorphometry characterization is an important procedure in describing neuronal cultures and identifying phenotypic differences. This task usually requires labor-intensive measurements and the classification of numerous neurites from large numbers of neurons in culture. To automate these measurements, we wrote AutoNeuriteJ, an imageJ/Fiji plugin that measures and classifies neurites from a very large number of neurons. We showed that AutoNeuriteJ is able to detect variations of neuritic growth induced by several compounds known to affect the neuronal growth. In these experiments measurement of more than 5000 mouse neurons per conditions was obtained within a few hours. Moreover, by analyzing mouse neurons deficient for the microtubule associated protein 6 (MAP6) and wild type neurons we illustrate that AutoNeuriteJ is capable to detect subtle phenotypic difference in axonal length. Overall the use of AutoNeuriteJ will provide rapid, unbiased and accurate measurement of neuron morphologies.

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Mutagenesis of theN-glycosylation site of hNaSi-1 reduces transport activity

AJP Cell Physiology ◽

10.1152/ajpcell.00162.2003 ◽

2003 ◽

Vol 285 (5) ◽

pp. C1188-C1196 ◽

Cited By ~ 25

Author(s):

Hongyan Li ◽

Ana M. Pajor

Keyword(s):

Xenopus Oocytes ◽

Polyclonal Antibodies ◽

Membrane Vesicles ◽

Glycosylation Site ◽

Transport Activity ◽

Wild Type ◽

Glutathione S Transferase ◽

Mutant Proteins ◽

Amino Acid Peptide

The human Na+-sulfate cotransporter (hNaSi-1) belongs to the SLC13 gene family, which also includes the high-affinity Na+-sulfate cotransporter (hSUT-1) and the Na+-dicarboxylate cotransporters (NaDC). In this study, the location and functional role of the N-glycosylation site of hNaSi-1 were studied using antifusion protein antibodies. Polyclonal antibodies against a glutathione S-transferase fusion protein containing a 65-amino acid peptide of hNaSi-1 (GST-Si65) were raised in rabbits, purified, and then used in Western blotting and immunofluorescence experiments. The antibodies recognized native NaSi-1 proteins in pig and rat brush-border membrane vesicles as well as the recombinant proteins expressed in Xenopus oocytes. Wild-type hNaSi-1 and two N-glycosylation site mutant proteins, N591Y and N591A, were functionally expressed and studied in Xenopus oocytes. The apparent mass of N591Y was not affected by treatment with peptide- N-glycosylase F, in contrast to the mass of wild-type hNaSi-1, which was reduced by up to 15 kDa, indicating that Asn591is the N-glycosylation site. Although the cell surface abundance of the two glycosylation site mutants, N591Y and N591A, was greater than that of wild-type hNaSi-1, both mutants had greatly reduced Vmax, with no change in Km. These results suggest that Asn591and/or N-glycosylation is critical for transport activity in NaSi-1.

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Investigation of the Stability of Yeast rad52 Mutant Proteins Uncovers Post-translational and Transcriptional Regulation of Rad52p

Genetics ◽

10.1093/genetics/163.1.91 ◽

2003 ◽

Vol 163 (1) ◽

pp. 91-101 ◽

Cited By ~ 1

Author(s):

Erin N Asleson ◽

Dennis M Livingston

Keyword(s):

Half Life ◽

Translational Regulation ◽

Cellular Level ◽

Missense Mutations ◽

Wild Type ◽

Mutant Proteins ◽

Gal1 Promoter ◽

Rad52 Protein ◽

The Stability ◽

Mutant Forms

Abstract We investigated the stability of the Saccharomyces cerevisiae Rad52 protein to learn how a cell controls its quantity and longevity. We measured the cellular levels of wild-type and mutant forms of Rad52p when expressed from the RAD52 promoter and the half-lives of the various forms of Rad52p when expressed from the GAL1 promoter. The wild-type protein has a half-life of 15 min. rad52 mutations variably affect the cellular levels of the protein products, and these levels correlate with the measured half-lives. While missense mutations in the N terminus of the protein drastically reduce the cellular levels of the mutant proteins, two mutations—one a deletion of amino acids 210-327 and the other a missense mutation of residue 235—increase the cellular level and half-life more than twofold. These results suggest that Rad52p is subject to post-translational regulation. Proteasomal mutations have no effect on Rad52p half-life but increase the amount of RAD52 message. In contrast to Rad52p, the half-life of Rad51p is >2 hr, and RAD51 expression is unaffected by proteasomal mutations. These differences between Rad52p and Rad51p suggest differential regulation of two proteins that interact in recombinational repair.

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Imbalance between Expression of FOXC2 and Its lncRNA in Lymphedema-Distichiasis Caused by Frameshift Mutations

Genes ◽

10.3390/genes12050650 ◽

2021 ◽

Vol 12 (5) ◽

pp. 650

Author(s):

Sara Missaglia ◽

Daniela Tavian ◽

Sandro Michelini ◽

Paolo Enrico Maltese ◽

Andrea Bonanomi ◽

...

Keyword(s):

Mrna Stability ◽

Healthy Subjects ◽

Lymphatic System ◽

System Development ◽

Bioinformatic Analysis ◽

Wild Type ◽

Mutant Proteins ◽

Frameshift Mutations ◽

Forkhead Box ◽

Frameshift Mutant

Forkhead-box C2 (FOXC2) is a transcription factor involved in lymphatic system development. FOXC2 mutations cause Lymphedema-distichiasis syndrome (LD). Recently, a natural antisense was identified, called lncRNA FOXC2-AS1, which increases FOXC2 mRNA stability. No studies have evaluated FOXC2 and FOXC2-AS1 blood expression in LD and healthy subjects. Here, we show that FOXC2 and FOXC-AS1 expression levels were similar in both controls and patients, and a significantly higher amount of both RNAs was observed in females. A positive correlation between FOXC2 and FOXC2-AS1 expression was found in both controls and patients, excluding those with frameshift mutations. In these patients, the FOXC2-AS1/FOXC2 ratio was about 1:1, while it was higher in controls and patients carrying other types of mutations. The overexpression or silencing of FOXC2-AS1 determined a significant increase or reduction in FOXC2 wild-type and frameshift mutant proteins, respectively. Moreover, confocal and bioinformatic analysis revealed that these variations caused the formation of nuclear proteins aggregates also involving DNA. In conclusion, patients with frameshift mutations presented lower values of the FOXC2-AS1/FOXC2 ratio, due to a decrease in FOXC2-AS1 expression. The imbalance between FOXC2 mRNA and its lncRNA could represent a molecular mechanism to reduce the amount of FOXC2 misfolded proteins, protecting cells from damage.

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Activation of Kinin B1R Upregulates ADAM17 and Results in ACE2 Shedding in Neurons

International Journal of Molecular Sciences ◽

10.3390/ijms22010145 ◽

2020 ◽

Vol 22 (1) ◽

pp. 145

Author(s):

Rohan Umesh Parekh ◽

Srinivas Sriramula

Keyword(s):

Protein Expression ◽

Renin Angiotensin System ◽

Neuronal Cultures ◽

Wild Type ◽

High Concentration ◽

Neurogenic Hypertension ◽

B1 Receptor ◽

Gene And Protein Expression ◽

Kinin B1 Receptor ◽

Specific Antagonist

Angiotensin converting enzyme 2 (ACE2) is a critical component of the compensatory axis of the renin angiotensin system. Alterations in ACE2 gene and protein expression, and activity mediated by A Disintegrin And Metalloprotease 17 (ADAM17), a member of the “A Disintegrin And Metalloprotease” (ADAM) family are implicated in several cardiovascular and neurodegenerative diseases. We previously reported that activation of kinin B1 receptor (B1R) in the brain increases neuroinflammation, oxidative stress and sympathoexcitation, leading to the development of neurogenic hypertension. We also showed evidence for ADAM17-mediated ACE2 shedding in neurons. However, whether kinin B1 receptor (B1R) activation has any role in altering ADAM17 activity and its effect on ACE2 shedding in neurons is not known. In this study, we tested the hypothesis that activation of B1R upregulates ADAM17 and results in ACE2 shedding in neurons. To test this hypothesis, we stimulated wild-type and B1R gene-deleted mouse neonatal primary hypothalamic neuronal cultures with a B1R-specific agonist and measured the activities of ADAM17 and ACE2 in neurons. B1R stimulation significantly increased ADAM17 activity and decreased ACE2 activity in wild-type neurons, while pretreatment with a B1R-specific antagonist, R715, reversed these changes. Stimulation with specific B1R agonist Lys-Des-Arg9-Bradykinin (LDABK) did not show any effect on ADAM17 or ACE2 activities in neurons with B1R gene deletion. These data suggest that B1R activation results in ADAM17-mediated ACE2 shedding in primary hypothalamic neurons. In addition, stimulation with high concentration of glutamate significantly increased B1R gene and protein expression, along with increased ADAM17 and decreased ACE2 activities in wild-type neurons. Pretreatment with B1R-specific antagonist R715 reversed these glutamate-induced effects suggesting that indeed B1R is involved in glutamate-mediated upregulation of ADAM17 activity and ACE2 shedding.

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Novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway

Scientific Reports ◽

10.1038/s41598-021-90952-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Safia Akhtar ◽

Silas A. Culver ◽

Helmy M. Siragy

Keyword(s):

Protein Expression ◽

High Fat Diet ◽

Normal Diet ◽

Receptor Expression ◽

Wild Type ◽

High Fat ◽

Renal Gluconeogenesis ◽

Mrna And Protein Expression ◽

Polymerase Chain ◽

Renal Expression

AbstractRecent studies suggested that renal gluconeogenesis is substantially stimulated in the kidney in presence of obesity. However, the mechanisms responsible for such stimulation are not well understood. Recently, our laboratory demonstrated that mice fed high fat diet (HFD) exhibited increase in renal Atp6ap2 [also known as (Pro)renin receptor] expression. We hypothesized that HFD upregulates renal gluconeogenesis via Atp6ap2-PGC-1α and AKT pathway. Using real-time polymerase chain reaction, western blot analysis and immunostaining, we evaluated renal expression of the Atp6ap2 and renal gluconeogenic enzymes, PEPCK and G6Pase, in wild type and inducible nephron specific Atp6ap2 knockout mice fed normal diet (ND, 12 kcal% fat) or a high-fat diet (HFD, 45 kcal% fat) for 8 weeks. Compared with ND, HFD mice had significantly higher body weight (23%) (P < 0.05), renal mRNA and protein expression of Atp6ap2 (39 and 35%), PEPCK (44 and 125%) and G6Pase (39 and 44%) respectively. In addition, compared to ND, HFD mice had increased renal protein expression of PGC-1α by 32% (P < 0.05) and downregulated AKT by 33% (P < 0.05) respectively in renal cortex. Atp6ap2-KO abrogated these changes in the mice fed HFD. In conclusion, we identified novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway.

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