scholarly journals Pathogenic Mechanisms Underlying Stargardt Macular Degeneration Linked to Mutations in the Transmembrane Domains of ABCA4

2020 ◽  
Author(s):  
Fabian A. Garces ◽  
Jessica F. Scortecci ◽  
Robert S. Molday
Keyword(s):  
Atpase Activity ◽  
Protein Misfolding ◽  
Substrate Binding ◽  
Transmembrane Domains ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Stargardt Disease ◽  
Photoreceptor Outer Segments ◽  
Novel Treatments ◽  
Stargardt Macular Degeneration

AbstractABCA4 is an ATP-binding cassette (ABC) transporter predominantly expressed in photoreceptors where it transports the substrate N-retinylidene-phosphatidylethanolamine across disc membranes thereby facilitating the clearance of retinal compounds from photoreceptor outer segments. Loss of function mutations in ABCA4 cause the accumulation of bisretinoids leading to Stargardt disease (STGD1) and other retinopathies. In this study, we examined the expression and functional properties of ABCA4 harboring disease-causing missense mutations in the two transmembrane domains (TMDs) of ABCA4. Our results indicate that these mutations lead to protein misfolding, loss in substrate binding, decreased ATPase activity or a combination of these properties. Additionally, we identified an arginine (R653) in transmembrane segment 2 of ABCA4 as a residue essential for substrate binding and substrate-stimulated ATPase activity. The expression and functional activity of the TMD variants correlate well with the severity of STGD1. Our studies provide a basis for developing and evaluating novel treatments for STGD1.

scholarly journals Transmembrane Polar Relay Drives the Allosteric Regulation for ABCG5/G8 Sterol Transporter

2020 ◽  
Vol 21 (22) ◽  
pp. 8747 ◽  
Author(s):  
Bala M. Xavier ◽  
Aiman A. Zein ◽  
Angelica Venes ◽  
Junmei Wang ◽  
Jyh-Yeuan Lee
Keyword(s):  
Atpase Activity ◽  
Signal Transmission ◽  
Transmembrane Domains ◽  
Water Soluble ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Charged Amino Acids ◽  
Cholesteryl Hemisuccinate

The heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely, a polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

scholarly journals Transmembrane polar relay drives the allosteric regulation for ABCG5/G8 sterol transporter

2020 ◽  
Author(s):  
Bala M. Xavier ◽  
Aiman A. Zein ◽  
Angelica Venes ◽  
Junmei Wang ◽  
Jyh-Yeuan Lee
Keyword(s):  
Atpase Activity ◽  
Signal Transmission ◽  
Transmembrane Domains ◽  
Water Soluble ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Charged Amino Acids ◽  
Cholesteryl Hemisuccinate

AbstractThe heterodimeric ATP-binding cassette (ABC) sterol transporter, ABCG5/G8, is responsible for the biliary and transintestinal secretion of cholesterol and dietary plant sterols. Missense mutations of ABCG5/G8 can cause sitosterolemia, a loss-of-function disorder characterized by plant sterol accumulation and premature atherosclerosis. A new molecular framework was recently established by a crystal structure of human ABCG5/G8 and reveals a network of polar and charged amino acids in the core of the transmembrane domains, namely polar relay. In this study, we utilize genetic variants to dissect the mechanistic role of this transmembrane polar relay in controlling ABCG5/G8 function. We demonstrated a sterol-coupled ATPase activity of ABCG5/G8 by cholesteryl hemisuccinate (CHS), a relatively water-soluble cholesterol memetic, and characterized CHS-coupled ATPase activity of three loss-of-function missense variants, R543S, E146Q, and A540F, which are respectively within, in contact with, and distant from the polar relay. The results established an in vitro phenotype of the loss-of-function and missense mutations of ABCG5/G8, showing significantly impaired ATPase activity and loss of energy sufficient to weaken the signal transmission from the transmembrane domains. Our data provide a biochemical evidence underlying the importance of the polar relay and its network in regulating the catalytic activity of ABCG5/G8 sterol transporter.

scholarly journals Functional Characterization of ABCA4 Missense Variants Linked to Stargardt Macular Degeneration

2020 ◽  
Vol 22 (1) ◽  
pp. 185
Author(s):  
Fabian A. Garces ◽  
Jessica F. Scortecci ◽  
Robert S. Molday
Keyword(s):  
Atpase Activity ◽  
Macular Degeneration ◽  
Substrate Binding ◽  
Functional Characterization ◽  
Autosomal Recessive Disorder ◽  
Missense Mutations ◽  
Culture Cells ◽  
Normal Expression ◽  
Class 1 ◽  
Stargardt Macular Degeneration

ABCA4 is an ATP-binding cassette (ABC) transporter expressed in photoreceptors, where it transports its substrate, N-retinylidene-phosphatidylethanolamine (N-Ret-PE), across outer segment membranes to facilitate the clearance of retinal from photoreceptors. Mutations in ABCA4 cause Stargardt macular degeneration (STGD1), an autosomal recessive disorder characterized by a loss of central vision and the accumulation of bisretinoid compounds. The purpose of this study was to determine the molecular properties of ABCA4 variants harboring disease-causing missense mutations in the transmembrane domains. Thirty-eight variants expressed in culture cells were analyzed for expression, ATPase activities, and substrate binding. On the basis of these properties, the variants were divided into three classes: Class 1 (severe variants) exhibited significantly reduced ABCA4 expression and basal ATPase activity that was not stimulated by its substrate N-Ret-PE; Class 2 (moderate variants) showed a partial reduction in expression and basal ATPase activity that was modestly stimulated by N-Ret-PE; and Class 3 (mild variants) displayed expression and functional properties comparable to normal ABCA4. The p.R653C variant displayed normal expression and basal ATPase activity, but lacked substrate binding and ATPase activation, suggesting that arginine 653 contributes to N-Ret-PE binding. Our classification provides a basis for better understanding genotype–phenotype correlations and evaluating therapeutic treatments for STGD1.

Loss of PERK Signaling Results in Impaired Granulopoiesis in Transgenic Mice Expressing Mutant Ela2.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 551-551
Author(s):  
Suparna Nanua ◽  
Jun Xia ◽  
Mark Murakami ◽  
Jill Woloszynek ◽  
Daniel C. Link
Keyword(s):  
Er Stress ◽  
Protein Misfolding ◽  
Conflicts Of Interest ◽  
Fetal Liver ◽  
Clinical Samples ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Granulocytic Differentiation ◽  
Disease Pathogenesis ◽  
Chemical Inducers

Abstract Abstract 551 Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis characterized by chronic neutropenia, a block in granulocytic differentiation at the promyelocyte/myelocyte stage, and a marked propensity to develop acute myeloid leukemia. Approximately 50% of cases of SCN are associated with germline heterozygous mutations of ELA2, encoding neutrophil elastase (NE). To date, 59 different, mostly missense, mutations of ELA2 have been reported. A unifying mechanism by which all of the different ELA2 mutants disrupt granulopoiesis is lacking. We and others previously proposed a model in which the ELA2 mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately apoptosis of granulocytic precursors. Testing this (and other) models has been limited by the rarity of SCN and difficulty in obtaining clinical samples for testing. We previously reported preliminary findings of a novel transgenic mouse expressing a truncation mutation of Ela2 (G193X) reproducing a similar mutation found in some patients with SCN (2008 ASH abstract #314). We showed that the G193X Ela2 allele produced the expected truncated protein that was rapidly degraded. Surprisingly, basal and stress granulopoiesis were normal. We hypothesized that reduced expression of Ela2 in murine compared with human granulocytic precursors resulted in less delivery of misfolded mutant NE protein to the ER, attenuating UPR activation and preserving granulopoiesis in G193X Ela2 mice. Consistent with this hypothesis, only modest evidence of UPR activation was observed in G193X Ela2 granulocytic precursors, and these cells displayed increased sensitivity to chemical inducers of ER stress compared with wildtype granulocytic precursors. The UPR model of disease pathogenesis predicts that inhibition of the cellular pathways that handle misfolded proteins may sensitize G193X Ela2 cells to ER stress and result in impaired granulocytic differentiation. To test this prediction, we crossed G193X Ela2 mice with mice lacking protein kinase RNA (PKR)-like ER kinase (PERK); PERK is one of three major ER-resident proteins that sense ER stress and activate the UPR. Of note, homozygous loss-of-function mutations of PERK (EIF2AK3) are responsible for Wolcott-Rallison syndrome, which is characterized by infantile diabetes and neutropenia in approximately 50% of cases. Since PERK deficiency is embryonic lethal, we transplanted fetal liver cells from PERK-/-, PERK-/- × G193X Ela2, and wild type embryos into irradiated recipients. Complete donor engraftment was observed in all cohorts. Basal granulopoiesis was normal in mice reconstituted with PERK-/- cells. However, in the PERK-/- × G193X Ela2 chimeras, though blood neutrophil counts were normal, a significant reduction in bone marrow neutrophils was observed [6.01 × 106/femur ± 0.92 (PERK-/-) versus 3.14 × 106 ± 0.88 (PERK-/- × G193X Ela2); p < 0.001]. These data show that loss of PERK signaling combined with G193X Ela2 expression results in impaired granulopoiesis, providing new evidence in support of the UPR model of disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Second-Generation Pharmacological Chaperones: Beyond Inhibitors

Molecules ◽  
2020 ◽  
Vol 25 (14) ◽  
pp. 3145 ◽  
Author(s):  
My Lan Tran ◽  
Yves Génisson ◽  
Stéphanie Ballereau ◽  
Cécile Dehoux
Keyword(s):  
Protein Misfolding ◽  
First Generation ◽  
Second Generation ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Pharmacological Chaperone ◽  
Conformational Diseases ◽  
Pharmacological Chaperones ◽  
Drug Candidates ◽  
Binding Pockets

Protein misfolding induced by missense mutations is the source of hundreds of conformational diseases. The cell quality control may eliminate nascent misfolded proteins, such as enzymes, and a pathological loss-of-function may result from their early degradation. Since the proof of concept in the 2000s, the bioinspired pharmacological chaperone therapy became a relevant low-molecular-weight compound strategy against conformational diseases. The first-generation pharmacological chaperones were competitive inhibitors of mutant enzymes. Counterintuitively, in binding to the active site, these inhibitors stabilize the proper folding of the mutated protein and partially rescue its cellular function. The main limitation of the first-generation pharmacological chaperones lies in the balance between enzyme activity enhancement and inhibition. Recent research efforts were directed towards the development of promising second-generation pharmacological chaperones. These non-inhibitory ligands, targeting previously unknown binding pockets, limit the risk of adverse enzymatic inhibition. Their pharmacophore identification is however challenging and likely requires a massive screening-based approach. This review focuses on second-generation chaperones designed to restore the cellular activity of misfolded enzymes. It intends to highlight, for a selected set of rare inherited metabolic disorders, the strategies implemented to identify and develop these pharmacologically relevant small organic molecules as potential drug candidates.

scholarly journals Cryo-EM structures of the ABCA4 importer reveal mechanisms underlying substrate binding and Stargardt disease

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jessica Fernandes Scortecci ◽  
Laurie L. Molday ◽  
Susan B. Curtis ◽  
Fabian A. Garces ◽  
Pankaj Panwar ◽  
...  
Keyword(s):  
Bound States ◽  
Vision Loss ◽  
Substrate Binding ◽  
Macular Dystrophy ◽  
Loss Of Function ◽  
Stargardt Disease ◽  
Binding Domains ◽  
Severe Vision Loss ◽  
Photoreceptor Membranes ◽  
Underlying Substrate

AbstractABCA4 is an ATP-binding cassette (ABC) transporter that flips N-retinylidene-phosphatidylethanolamine (N-Ret-PE) from the lumen to the cytoplasmic leaflet of photoreceptor membranes. Loss-of-function mutations cause Stargardt disease (STGD1), a macular dystrophy associated with severe vision loss. To define the mechanisms underlying substrate binding and STGD1, we determine the cryo-EM structure of ABCA4 in its substrate-free and bound states. The two structures are similar and delineate an elongated protein with the two transmembrane domains (TMD) forming an outward facing conformation, extended and twisted exocytoplasmic domains (ECD), and closely opposed nucleotide binding domains. N-Ret-PE is wedged between the two TMDs and a loop from ECD1 within the lumen leaflet consistent with a lateral access mechanism and is stabilized through hydrophobic and ionic interactions with residues from the TMDs and ECDs. Our studies provide a framework for further elucidating the molecular mechanism associated with lipid transport and disease and developing promising disease interventions.

scholarly journals Alterations in ZnT1 expression and function lead to impaired intracellular zinc homeostasis in cancer

2019 ◽  
Vol 5 (1) ◽  
Author(s):  
Adrian Israel Lehvy ◽  
Guy Horev ◽  
Yarden Golan ◽  
Fabian Glaser ◽  
Yael Shammai ◽  
...  
Keyword(s):  
Malignant Tumors ◽  
Zinc Homeostasis ◽  
Healthy Population ◽  
Missense Mutations ◽  
Protein Alignment ◽  
Loss Of Function ◽  
Intracellular Zinc ◽  
Dismal Prognosis ◽  
Zinc Levels ◽  
And Function

Abstract Zinc is vital for the structure and function of ~3000 human proteins and hence plays key physiological roles. Consequently, impaired zinc homeostasis is associated with various human diseases including cancer. Intracellular zinc levels are tightly regulated by two families of zinc transporters: ZIPs and ZnTs; ZIPs import zinc into the cytosol from the extracellular milieu, or from the lumen of organelles into the cytoplasm. In contrast, the vast majority of ZnTs compartmentalize zinc within organelles, whereas the ubiquitously expressed ZnT1 is the sole zinc exporter. Herein, we explored the hypothesis that qualitative and quantitative alterations in ZnT1 activity impair cellular zinc homeostasis in cancer. Towards this end, we first used bioinformatics to analyze inactivating mutations in ZIPs and ZNTs, catalogued in the COSMIC and gnomAD databases, representing tumor specimens and healthy population controls, respectively. ZnT1, ZnT10, ZIP8, and ZIP10 showed extremely high rates of loss of function mutations in cancer as compared to healthy controls. Analysis of the putative functional impact of missense mutations in ZnT1-ZnT10 and ZIP1-ZIP14, using homologous protein alignment and structural predictions, revealed that ZnT1 displays a markedly increased frequency of predicted functionally deleterious mutations in malignant tumors, as compared to a healthy population. Furthermore, examination of ZnT1 expression in 30 cancer types in the TCGA database revealed five tumor types with significant ZnT1 overexpression, which predicted dismal prognosis for cancer patient survival. Novel functional zinc transport assays, which allowed for the indirect measurement of cytosolic zinc levels, established that wild type ZnT1 overexpression results in low intracellular zinc levels. In contrast, overexpression of predicted deleterious ZnT1 missense mutations did not reduce intracellular zinc levels, validating eight missense mutations as loss of function (LoF) mutations. Thus, alterations in ZnT1 expression and LoF mutations in ZnT1 provide a molecular mechanism for impaired zinc homeostasis in cancer formation and/or progression.

scholarly journals The Pah-R261Q mouse reveals oxidative stress associated with amyloid-like hepatic aggregation of mutant phenylalanine hydroxylase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Oscar Aubi ◽  
Karina S. Prestegård ◽  
Kunwar Jung-KC ◽  
Tie-Jun Sten Shi ◽  
Ming Ying ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Protein Misfolding ◽  
Phenylalanine Hydroxylase ◽  
Hepatic Tissue ◽  
Loss Of Function ◽  
Advantageous Position ◽  
Protein Misfolding And Aggregation ◽  
Activity Decrease ◽  
Systemic Accumulation ◽  
Altered Lipid

AbstractPhenylketonuria (PKU) is caused by autosomal recessive variants in phenylalanine hydroxylase (PAH), leading to systemic accumulation of L-phenylalanine (L-Phe) that may reach neurotoxic levels. A homozygous Pah-R261Q mouse, with a highly prevalent misfolding variant in humans, reveals the expected hepatic PAH activity decrease, systemic L-Phe increase, L-tyrosine and L-tryptophan decrease, and tetrahydrobiopterin-responsive hyperphenylalaninemia. Pah-R261Q mice also present unexpected traits, including altered lipid metabolism, reduction of liver tetrahydrobiopterin content, and a metabolic profile indicative of oxidative stress. Pah-R261Q hepatic tissue exhibits large ubiquitin-positive, amyloid-like oligomeric aggregates of mutant PAH that colocalize with selective autophagy markers. Together, these findings reveal that PKU, customarily considered a loss-of-function disorder, can also have toxic gain-of-function contribution from protein misfolding and aggregation. The proteostasis defect and concomitant oxidative stress may explain the prevalence of comorbid conditions in adult PKU patients, placing this mouse model in an advantageous position for the discovery of mutation-specific biomarkers and therapies.

scholarly journals MBRS-17. EXAMINING THE ROLE OF LHX9 IN GROUP 3 MEDULLOBLASTOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii401-iii401
Author(s):  
Sarah Injac ◽  
L Frank Huang ◽  
Stephen Mack ◽  
Frank Braun ◽  
Yuchen Du ◽  
...  
Keyword(s):  
Drug Repositioning ◽  
Single Agent ◽  
Xenograft Model ◽  
Cell Killing ◽  
Rna Seq ◽  
Loss Of Function ◽  
Novel Treatments ◽  
Novel Approach ◽  
Group 3

Abstract Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Despite major advances in our understanding of the biology of MB, novel treatments remain urgently needed. Using a chemical-genomics driven drug repositioning strategy, we identified the cardiac glycoside family of compounds as potential treatments for Group 3 MB. We subsequently demonstrated that single-agent treatment with digoxin prolongs survival in a patient-derived xenograft model (PDOX) of Group 3 MB to a degree comparable to radiation therapy, a mainstay in the treatment of MB. Finally, we examined the mechanism of digoxin-mediated cell killing using RNA-seq. This work identified LHX9, a member of the LIM homeobox family of transcription factors, as the gene most significantly down-regulated following treatment (Huang and Injac et al, Sci Trans Medicine, 2018). Homologs of LHX9 play key roles in cerebellar development via spatially and temporally restricted expression and LHX9 has been proposed as a core transcription factor (TF) in the regulatory circuitry of Group 3 tumors. Loss of function of other core TFs has been shown to impact MB growth. The role of LHX9 in MB, however, has not been previously experimentally evaluated. We now report that knockdown of LHX9 in MB-derived cell lines results in marked growth inhibition raising the possibility that loss of LHX9 plays a major role in digoxin-mediated cell killing and that LHX9 represents a key dependency required for the growth of Group 3 MB. Clinical targeting of core TFs would represent a novel approach to targeting this devastating disease.

Abstract 18: Synoviolin, an E3 Ubiquitin Ligase, Modulates Cardiac Myocyte Size and Restores Heart Function in Hypertrophic Cardiomyopathy

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Shirin Doroudgar ◽  
Mirko Völkers ◽  
Donna J Thuerauf ◽  
Ashley Bumbar ◽  
Mohsin Khan ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Misfolding ◽  
Cardiac Myocyte ◽  
Protein Quality ◽  
Heart Function ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Calcium Handling ◽  
Loss Of Function

The endoplasmic reticulum (ER) is essential for protein homeostasis, or proteostasis, which governs the balance of the proteome. In addition to secreted and membrane proteins, proteins bound for many other cellular locations are also made on ER-bound ribosomes, emphasizing the importance of protein quality and quantity control in the ER. Unlike cytosolic E3 ubiquitin ligases studied in the heart, synoviolin/Hrd1, which has not been studied in the heart, is an ER transmembrane E3 ubiquitin ligase, which we found to be upregulated upon protein misfolding in cardiac myocytes. Given the strategic location of synoviolin in the ER membrane, we addressed the hypothesis that synoviolin is critical for regulating the balance of the proteome, and accordingly, myocyte size. We showed that in vitro, adenovirus-mediated overexpression of synoviolin decreased cardiac myocyte size and protein synthesis, but unlike atrophy-related ubiquitin ligases, synoviolin did not increase global protein degradation. Furthermore, targeted gene therapy using adeno-associated virus 9 (AAV9) showed that overexpression of synoviolin in the left ventricle attenuated maladaptive cardiac hypertrophy and preserved cardiac function in mice subjected to trans-aortic constriction (AAV9-control TAC = 22.5 ± 6.2% decrease in EF vs. AAV9-synoviolin TAC at 6 weeks post TAC; P<0.001), and decreased mTOR activity. Since calcium is a major regulator of cardiac myocyte size, we examined the effects of synoviolin gain- or loss-of-function, using AAV9-synoviolin, or an miRNA designed to knock down synoviolin, respectively. While synoviolin gain-of-function did not affect calcium handling in isolated adult myocytes, synoviolin loss-of-function increased calcium transient amplitude (P<0.01), prolonged spark duration (P<0.001), and increased spark width (P<0.001). Spark frequency and amplitude were unaltered upon synoviolin gain- or loss-of-function. Whereas SR calcium load was unaltered by synoviolin loss-of-function, SERCA-mediated calcium removal was reduced (P<0.05). In conclusion, our studies suggest that in the heart, synoviolin is 1) a critical component of proteostasis, 2) a novel determinant of cardiac myocyte size, and 3) necessary for proper calcium handling.

Sign in / Sign up

Export Citation Format

Share Document