Inna Sabirzhanova | University of Maryland Baltimore (original) (raw)

Papers by Inna Sabirzhanova

Δ27–264-CFTR was transfected into HEK 293 cell lines stably expressing the mutants. Δ27–... more Δ27–264-CFTR was transfected into HEK 293 cell lines stably expressing the mutants. Δ27–264-is able to increase significantly band C of (A) N1303K and (B) S1235R. Graphs show that the fully glycosylated protein is increased by 3 times for (C) N1303K and more than 2 times for (D) S1235R. Cells were cultured for 48 hours at 37°C with or without the truncated construct (Δ27–264). Data are expressed as the mean ± SD of 3 independent experiments.</p

(A) Samples were used for western blot analysis and blotted with the ... more (A) Samples were used for western blot analysis and blotted with the various antibodies as specified in the figure. (B) The main difference in the total lysate is a significant increase in the total amount of HSP27 for S1235R and a significant but slightly decreased binding of HSP27 to N1303K when compared with the control (HEK 293 cell lines without any CFTR mutation). The graphs show that HSP27 in the cell line bearing the S1235R mutant, whether treated or untreated with the compounds, is four times higher than in the control and the N1303K cell line. No differences were found for all the other tested proteins (HSP40, 70, 90, VCP and HDAC6). No differences were found for HSPs, VCP and HDAC6 between the mutations alone or treated with compounds C3 + C4. Cells were cultured for 16 hours at 37°C with or without correctors. Data are expressed as the mean ± SD of 3 independent experiments</p

An HEK 293 cell line stably expressing N1303K was treated with (A) th... more An HEK 293 cell line stably expressing N1303K was treated with (A) the lysosome inhibitor E64, (B) proteasome inhibitor MG132, or (C) aggresome inhibitor tubacin. (D) The graph shows that the N1303K mutant is mainly degraded by the proteasome and aggresome; no effect was detected after lysosome treatment. Data are expressed as the mean ± SD of 3 independent experiments.</p

Investigative Ophthalmology & Visual Science, 2015

Investigative Ophthalmology & Visual Science, 2018

Journal of Cystic Fibrosis, 2018

The missing phenylalanine at position 508, located in nucleotide-binding domain (NBD1) of the cys... more The missing phenylalanine at position 508, located in nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane regulator (CFTR), is the most common cystic fibrosis mutation. Severe disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we used a combination of biochemical, cell biological and electrophysiological approaches and newly created cell lines to study two disease-causing NBD2 mutants, N1303K and S1235R. We observed that neither was sensitive to E64, a cysteine protease inhibitor. However, further investigation showed that when treated with a combination of correctors, C4 + C18, both mutants also responded to E64. Further exploration to assess aggresome throughput using the autophagy regulator LC3 as a marker showed that, in the absence of correctors, N1303K showed a stalled throughput of LC3-II to the aggresome. The throughput became active again after treatment with the corrector combination C4 + C18. Confocal microscopic studies showed that the N1303K and S1235R mutant proteins both co-localized with LC3, but this co-localization was abolished by the corrector combination and, to a lesser extent, by VX-809. Both the corrector combination and VX-809 increased the CFTR chloride channel function of both mutants. We conclude that correctors have a dual effect, particularly on N1303K: they improve trafficking and function at the plasma membrane and reduce the association with autophagosomes. After treatment with correctors persistent degradation by the autophagosome may limit restoration of function. Thus, mutations in NBD2 of CFTR, in contrast to ΔF508-CFTR, may require additional personalized strategies to rescue them.

Cellular Physiology and Biochemistry, 2019

This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 I... more This article is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND). Usage and distribution for commercial purposes as well as any distribution of modified material requires written permission.

Cellular Physiology and Biochemistry, 2019

Background/Aims: Because of the small size of adeno-associated virus, AAV, the cystic fibrosis co... more Background/Aims: Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via transcomplementation. The purpose of this study is to shed light on where in the cell transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR. Methods: We treated CF airway cells (CFBE41o-) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports. Results: We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that transcomplementation occurs through a bimolecular interaction. We found that transcomplementation could increase the Isc in CFBE41ocells stably expressing additional wt-CFTR or F508-del and in parental CFBE41ocells expressing endogenous levels of F508-del. Conclusion: We conclude that the functional rescue of F508-del by transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients.

Cellular Physiology and Biochemistry, 2018

Background/Aims: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the C... more Background/Aims: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR). ΔF508, the most common mutation, is a misfolded protein that is retained in the endoplasmic reticulum and degraded, precluding delivery to the cell surface [1]. Methods: Here we use a combination of western blotting, immunoprecipitation, and short circuit current techniques combined with confocal microscopy to address whether the SNARE attachment protein, STX8 plays a role in ΔF508’s processing and movement out of the ER. Results: Although the SNARE protein STX8 is thought to be functionally related and primarily localized to early endosomes, we show that silencing of STX8, particularly in the presence of the Vertex corrector molecule C18, rescues ΔF508-CFTR, allowing it to reach the cell surface and increasing CFTR-dependent chloride currents by approximately 2.5-fold over control values. STX8 silencing reduced the binding of quality control...

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 2018

The CFTR-Associated Ligand (CAL), a PDZ domain containing protein with two coiled-coil domains, r... more The CFTR-Associated Ligand (CAL), a PDZ domain containing protein with two coiled-coil domains, reduces cell surface WT CFTR through degradation in the lysosome by a well-characterized mechanism. However, CAL's regulatory effect on ΔF508 CFTR has remained almost entirely uninvestigated. In this study, we describe a previously unknown pathway for CAL by which it regulates the membrane expression of ΔF508 CFTR through arrest of ΔF508 CFTR trafficking in the endoplasmic reticulum (ER) using a combination of cell biology, biochemistry and electrophysiology. We demonstrate that CAL is an ER localized protein that binds to ΔF508 CFTR and is degraded in the 26S proteasome. When CAL is inhibited, ΔF508 CFTR retention in the ER decreases and cell surface expression of mature functional ΔF508 CFTR is observed alongside of enhanced expression of plasma membrane scaffolding protein NHERF1. Chaperone proteins regulate this novel process, and ΔF508 CFTR binding to HSP40, HSP90, HSP70, VCP, an...

Cellular Physiology and Biochemistry, 2017

Background/Aims: Premature degradation of mutated cystic fibrosis transmembrane conductance regul... more Background/Aims: Premature degradation of mutated cystic fibrosis transmembrane conductance regulator (CFTR) protein causes cystic fibrosis (CF), the commonest Mendelian disease in Caucasians. Despite recent advances in precision medicines for CF patients, many CFTR mutants have not been characterized and the effects of these new therapeutic approaches are still unclear for those mutants. Methods: Cells transfected or stably expressing four CFTR transmembrane-domain mutants (G85E, E92K, L1077P, and M1101K) were used to: 1) characterize the mutants according to their protein expression, thermal sensitivity, and degradation pathways; 2) evaluate the effects of correctors in rescuing them; and 3) explore the effects of correctors on CFTR interactions with proteostasis components. Results: All four mutants exhibited lower protein expression than did wild type-CFTR, and they were degraded by proteasomes and aggresomes. At low temperature, only cells expressing the mutants L1077P and M110...

Journal of Cystic Fibrosis, 2016

Stop codon mutations are common in CF. To address if function can be restored by transcomplementa... more Stop codon mutations are common in CF. To address if function can be restored by transcomplementation we infected CFBE41o-cell lines containing either R1162X or W1282X with AAV1 containing D264, or D27-264 or CFTRDR-missing residues 708-759. We found that transcomplementation with D264 or D27-264 generated CFTR currents that were approximately 2 fold greater for R1162X and 1.5 fold for W1282X compared to control. DRD transduction generated 3.5 and 4.5 more current than control for R1162X and W1282X, respectively. Conclusion: R1162X and R1282X can be rescued to a small extent with transcomplementation. However, the better option will most likely be achieved that contain AAV1-CFTR vectors which generates currents on their own.

Journal of Biological Chemistry, 2015

Background: Mutations in nucleotide binding domain 1 of ABCA4 cause Stargardt Disease. Results: C... more Background: Mutations in nucleotide binding domain 1 of ABCA4 cause Stargardt Disease. Results: Correctors rescue trafficking of NBD1 mutants by altering a proteostatic network of quality control proteins. Conclusion: Rescue of trafficking ABCA 4 mutants can be accomplished by correctors similar to CFTR. Significance: There is currently no treatment for Stargardt macular degeneration. Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment. Stargardt macular degeneration is the most common form of early onset macular degeneration, causing poor visual outcome (32). The prevalence is ϳ1 in 10,000 (23). Individuals with this disorder suffer from a loss of central vision and impaired dark adaptation due to progressive accumulation of lipofuscin that results in dysfunction of the retinal pigmented epithelium (RPE) 3 and photoreceptors (24). Mutations in ABCA4 are associated with Stargardt disease (17). In addition to being causative * This work was funded by the National Council for Scientific and Technological Development (CNPq) Brazil (Lopes Pacheco) and DK072084 (to W. B. G.). The authors declare that they have no conflicts of interest with the contents of this article.

Investigative Ophthalmology & Visual Science, 2015

Investigative Ophthalmology & Visual Science, 2018

Journal of Cystic Fibrosis, 2018

Cellular Physiology and Biochemistry, 2019

Cellular Physiology and Biochemistry, 2018

Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology, 2018

Cellular Physiology and Biochemistry, 2017

Journal of Cystic Fibrosis, 2016

Journal of Biological Chemistry, 2015