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1.
Redox Biol ; 37: 101739, 2020 10.
Article in English | MEDLINE | ID: mdl-33242767

ABSTRACT

Redox homeostasis regulates key cellular signaling in both physiology and pathology. While perturbations result in shifting the redox homeostasis towards oxidative stress are well documented, the influence of reductive stress (RS) in neurodegenerative diseases and its mechanisms are unknown. Here, we postulate that a redox shift towards the reductive arm (through the activation of Nrf2 signaling) will damage neurons and impair neurogenesis. In proliferating and differentiating neuroblastoma (Neuro 2a/N2a) cells, sulforaphane-mediated Nrf2 activation resulted in increased transcription/translation of antioxidants and glutathione (GSH) production along with significantly declined ROS in a dose-dependent manner leading to a reductive-redox state (i.e. RS). Interestingly, this resulted in endoplasmic reticulum (ER) stress leading to subsequent protein aggregation/proteotoxicity in neuroblastoma cells. Under RS, we also observed elevated Tau/α-synuclein and their co-localization with other protein aggregates in these cells. Surprisingly, we noticed that acute RS impaired neurogenesis as evidenced from reduced neurite outgrowth/length. Furthermore, maintaining the cells in a sustained RS condition (for five consecutive generations) dramatically reduced their differentiation and prevented the formation of axons (p < 0.05). This impairment in RS mediated neurogenesis occurs through the alteration of Tau dynamics i.e. RS activates the pathogenic GSK3ß/Tau cascade thereby promoting the phosphorylation of Tau leading to proteotoxicity. Of note, intermittent withdrawal of sulforaphane from these cells suppressed the proteotoxic insult and re-activated the differentiation process. Overall, this results suggest that either acute or chronic RS could hamper neurogenesis through GSK3ß/TAU signaling and proteotoxicity. Therefore, investigations identifying novel redox mechanisms impacting proteostasis are crucial to preserve neuronal health.


Subject(s)
Oxidative Stress , Protein Aggregates , Endoplasmic Reticulum Stress , Neurogenesis , Oxidation-Reduction
2.
Mol Cancer Ther ; 15(7): 1485-1494, 2016 07.
Article in English | MEDLINE | ID: mdl-27196775

ABSTRACT

Standard therapies used for the treatment of acute myeloid leukemia (AML) are cytotoxic agents that target rapidly proliferating cells. Unfortunately, this therapeutic approach has limited efficacy and significant toxicity and the majority of AML patients still die of their disease. In contrast to the poor prognosis of most AML patients, most individuals with a rare subtype of AML, acute promyelocytic leukemia, can be cured by differentiation therapy using regimens containing all-trans retinoic acid. GSK3 has been previously identified as a therapeutic target in AML where its inhibition can lead to the differentiation and growth arrest of leukemic cells. Unfortunately, existing GSK3 inhibitors lead to suboptimal differentiation activity making them less useful as clinical AML differentiation agents. Here, we describe the discovery of a novel GSK3 inhibitor, GS87. GS87 was discovered in efforts to optimize GSK3 inhibition for AML differentiation activity. Despite GS87's dramatic ability to induce AML differentiation, kinase profiling reveals its high specificity in targeting GSK3 as compared with other kinases. GS87 demonstrates high efficacy in a mouse AML model system and unlike current AML therapeutics, exhibits little effect on normal bone marrow cells. GS87 induces potent differentiation by more effectively activating GSK3-dependent signaling components including MAPK signaling as compared with other GSK3 inhibitors. GS87 is a novel GSK3 inhibitor with therapeutic potential as a differentiation agent for non-promyelocytic AML. Mol Cancer Ther; 15(7); 1485-94. ©2016 AACR.


Subject(s)
Antineoplastic Agents/pharmacology , Cell Differentiation/drug effects , Enzyme Inhibitors/pharmacology , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3/metabolism , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Animals , Biomarkers , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Female , Humans , Leukemia, Myeloid, Acute/drug therapy , MAP Kinase Signaling System/drug effects , Mice , Mice, Knockout , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Xenograft Model Antitumor Assays
3.
FASEB J ; 30(5): 1865-79, 2016 05.
Article in English | MEDLINE | ID: mdl-26839378

ABSTRACT

Recently we have reported that age-dependent decline in antioxidant levels accelerated apoptosis and skeletal muscle degeneration. Here, we demonstrate genetic ablation of the master cytoprotective transcription factor, nuclear factor (erythroid-derived-2)-like 2 (Nrf2), aggravates cardiotoxin (CTX)-induced tibialis anterior (TA) muscle damage. Disruption of Nrf2 signaling sustained the CTX-induced burden of reactive oxygen species together with compromised expression of antioxidant genes and proteins. Transcript/protein expression of phenotypic markers of muscle differentiation, namely paired box 7 (satellite cell) and early myogenic differentiation and terminal differentiation (myogenin and myosin heavy chain 2) were increased on d 2 and 4 postinjury but later returned to baseline levels on d 8 and 15 in wild-type (WT) mice. In contrast, these responses were persistently augmented in Nrf2-null mice suggesting that regulation of the regeneration-related signaling mechanisms require Nrf2 for normal functioning. Furthermore, Nrf2-null mice displayed slower regeneration marked by dysregulation of embryonic myosin heavy chain temporal expression. Histologic observations illustrated that Nrf2-null mice displayed smaller, immature TA muscle fibers compared with WT counterparts on d 15 after CTX injury. Improvement in TA muscle morphology and gain in muscle mass evident in the WT mice was not noticeable in the Nrf2-null animals. Taken together these data show that the satellite cell activation, proliferation, and differentiation requires a functional Nrf2 system for effective healing following injury.-Shelar, S. B., Narasimhan, M., Shanmugam, G., Litovsky, S. H., Gounder, S. S., Karan, G., Arulvasu, C., Kensler, T. W., Hoidal, J. R., Darley-Usmar, V. M., Rajasekaran, N. S. Disruption of nuclear factor (erythroid-derived-2)-like 2 antioxidant signaling: a mechanism for impaired activation of stem cells and delayed regeneration of skeletal muscle.


Subject(s)
Antioxidants/physiology , Cardiotoxins/toxicity , Muscle, Skeletal/drug effects , Muscle, Skeletal/growth & development , NF-E2-Related Factor 2/metabolism , Signal Transduction/physiology , Aging , Animals , Cell Line , Cell Proliferation/physiology , Gene Expression Regulation/physiology , Humans , Mice , Mice, Knockout , NF-E2-Related Factor 2/genetics , Regeneration/physiology , Satellite Cells, Skeletal Muscle/physiology , Stem Cells
4.
Mol Cancer Ther ; 15(4): 574-582, 2016 04.
Article in English | MEDLINE | ID: mdl-26883273

ABSTRACT

Inactivation of the p53 tumor suppressor by mutation or overexpression of negative regulators occurs frequently in cancer. As p53 plays a key role in regulating proliferation or apoptosis in response to DNA-damaging chemotherapies, strategies aimed at reactivating p53 are increasingly being sought. Strategies to reactivate wild-type p53 include the use of small molecules capable of releasing wild-type p53 from key, cellular negative regulators, such as Hdm2 and HdmX. Derivatives of the Hdm2 antagonist Nutlin-3 are in clinical trials. However, Nutlin-3 specifically disrupts Hdm2-p53, leaving tumors harboring high levels of HdmX resistant to Nutlin-3 treatment. Here, we identify CTX1, a novel small molecule that overcomes HdmX-mediated p53 repression. CTX1 binds directly to HdmX to prevent p53-HdmX complex formation, resulting in the rapid induction of p53 in a DNA damage-independent manner. Treatment of a panel of cancer cells with CTX1 induced apoptosis or suppressed proliferation and, importantly, CTX1 demonstrates promising activity as a single agent in a mouse model of circulating primary human leukemia. CTX1 is a small molecule HdmX inhibitor that demonstrates promise as a cancer therapeutic candidate. Mol Cancer Ther; 15(4); 574-82. ©2016 AACR.


Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Cycle Proteins , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , DNA Damage , Humans , Nuclear Proteins/antagonists & inhibitors , Protein Binding/drug effects , Proto-Oncogene Proteins/antagonists & inhibitors , Tumor Suppressor Protein p53/antagonists & inhibitors
5.
Dev Cell ; 31(5): 539-41, 2014 Dec 08.
Article in English | MEDLINE | ID: mdl-25490265

ABSTRACT

In this issue, Comai et al. present evidence that previous support for Myf5-independent myogenic cell lineages was confounded by inefficiencies in lineage marking and ablation. Here, Haldar et al. discuss other possible explanations for the inconsistencies between different data sets and reiterate their views on Myf5-independent myogenesis.


Subject(s)
Cell Lineage , DNA-Binding Proteins/metabolism , Muscle Proteins/metabolism , Muscle, Skeletal/cytology , Myogenic Regulatory Factor 5/metabolism , Animals
6.
Biochemistry ; 47(32): 8335-41, 2008 Aug 12.
Article in English | MEDLINE | ID: mdl-18636749

ABSTRACT

Both germinal and somatic isoforms of ACE are type I ectoproteins expressed on the cell surface from where the enzymatically active ectodomains are released to circulation by a regulated cleavage-secretion process. Our previous studies have shown that ACE-secretase activity is regulated by the ACE distal ectodomain and not by sequences at or around the cleavage site. In the current study we have identified that the ACE residues encompassing 343 to 655 of the germinal form are needed for its cleavage-secretion. To narrow down this region further, we have examined the cleavage-secretion of ACE-CD4 chimeric proteins in mammalian cells and Pichia pastoris. These experiments identified five residues (HGEKL) in the ACE region of the chimeric proteins that were essential for their cleavage-secretion. When the corresponding residues were substituted by alanine in native germinal and somatic ACE, the mutant proteins were not cleaved, although they were displayed on the cell surface and enzymatically active. These results demonstrated that a small region in the ectodomain of ACE is required for its cleavage at the juxtamembrane domain. This conclusion was further supported by our observation that secreted ACE inhibited cell-bound ACE cleavage-secretion, although the secreted form did not contain the cleavage site.


Subject(s)
Amino Acid Motifs/physiology , Cell Membrane/enzymology , Cell Membrane/metabolism , Peptidyl-Dipeptidase A/chemistry , Peptidyl-Dipeptidase A/metabolism , Amino Acid Motifs/genetics , Amino Acid Sequence , Animals , Cell Membrane/genetics , HeLa Cells , Humans , Lung/cytology , Lung/enzymology , Molecular Sequence Data , Peptidyl-Dipeptidase A/genetics , Pichia/enzymology , Pichia/genetics , Protein Structure, Tertiary/genetics , Rabbits
7.
J Clin Invest ; 118(8): 2908-16, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18654668

ABSTRACT

Familial macular degeneration is a clinically and genetically heterogeneous group of disorders characterized by progressive central vision loss. Here we show that an R373C missense mutation in the prominin 1 gene (PROM1) causes 3 forms of autosomal-dominant macular degeneration. In transgenic mice expressing R373C mutant human PROM1, both mutant and endogenous PROM1 were found throughout the layers of the photoreceptors, rather than at the base of the photoreceptor outer segments, where PROM1 is normally localized. Moreover, the outer segment disk membranes were greatly overgrown and misoriented, indicating defective disk morphogenesis. Immunoprecipitation studies showed that PROM1 interacted with protocadherin 21 (PCDH21), a photoreceptor-specific cadherin, and with actin filaments, both of which play critical roles in disk membrane morphogenesis. Collectively, our results identify what we believe to be a novel complex involved in photoreceptor disk morphogenesis and indicate a possible role for PROM1 and PCDH21 in macular degeneration.


Subject(s)
Antigens, CD/genetics , Glycoproteins/genetics , Macular Degeneration/genetics , Mutation, Missense , Peptides/genetics , Photoreceptor Cells, Vertebrate/metabolism , AC133 Antigen , Actin Cytoskeleton/metabolism , Animals , Antigens, CD/metabolism , Cadherin Related Proteins , Cadherins/metabolism , Electroretinography , Glycoproteins/metabolism , Humans , Macular Degeneration/physiopathology , Mice , Mice, Transgenic , Morphogenesis , Nerve Tissue Proteins/metabolism , Peptides/metabolism , Photoreceptor Cells, Vertebrate/ultrastructure
8.
Dev Cell ; 14(3): 437-45, 2008 Mar.
Article in English | MEDLINE | ID: mdl-18331721

ABSTRACT

In skeletal muscle development, the myogenic regulatory factors myf5 and myoD play redundant roles in the specification and maintenance of myoblasts, whereas myf6 has a downstream role in differentiating myocytes and myofibers. It is not clear whether the redundancy between myf5 and myoD is within the same cell lineage or between distinct lineages. Using lineage tracing and conditional cell ablation in mice, we demonstrate the existence of two distinct lineages in myogenesis: a myf5 lineage and a myf5-independent lineage. Ablating the myf5 lineage is compatible with myogenesis sustained by myf5-independent, myoD-expressing myoblasts, whereas ablation of the myf6 lineage leads to an absence of all differentiated myofibers, although early myogenesis appears to be unaffected. We also demonstrate here the existence of a significant myf5 lineage within ribs that has an important role in rib development, suggested by severe rib defects upon ablating the myf5 lineage.


Subject(s)
Muscle Development/physiology , Myoblasts, Skeletal/cytology , Myoblasts, Skeletal/metabolism , Myogenic Regulatory Factor 5/metabolism , Alleles , Animals , Gene Targeting , Mice , Mice, Knockout , Mice, Transgenic , Muscle Development/genetics , MyoD Protein/genetics , MyoD Protein/metabolism , Myoblasts, Skeletal/classification , Myogenic Regulatory Factor 5/deficiency , Myogenic Regulatory Factor 5/genetics , Myogenic Regulatory Factors/genetics , Myogenic Regulatory Factors/metabolism , Ribs/embryology , Ribs/metabolism
9.
Vision Res ; 47(5): 714-22, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17254625

ABSTRACT

ELOVL4 was first identified as a disease-causing gene in Stargardt macular dystrophy (STGD3, MIM 600110.) To date, three ELOVL4 mutations have been identified, all of which result in truncated proteins which induce autosomal dominant juvenile macular degenerations. Based on sequence homology, ELOVL4 is thought to be another member within a family of proteins functioning in the elongation of long chain fatty acids. However, the normal function of ELOVL4 is unclear. We generated Elovl4 knockout mice to determine if Elovl4 loss affects retinal development or function. Here we show that Elovl4 knockout mice, while perinatal lethal, exhibit normal retinal development prior to death at day of birth. Further, postnatal retinal development in Elovl4 heterozygous mice appears normal. Therefore haploinsufficiency for wildtype ELOVL4 in autosomal dominant macular degeneration likely does not contribute to juvenile macular degeneration in STGD3 patients. We found, however, that Elovl4+/- mice exhibit enhanced ERG scotopic and photopic a and b waves relative to wildtype Elovl4+/+ mice suggesting that reduced Elovl4 levels may impact retinal electrophysiological responses.


Subject(s)
Eye Proteins/genetics , Macular Degeneration/genetics , Membrane Proteins/genetics , Adult , Animals , Disease Models, Animal , Electroretinography , Erythrocyte Membrane/chemistry , Eye Proteins/metabolism , Eye Proteins/physiology , Fatty Acids/blood , Haplotypes , Humans , Macular Degeneration/pathology , Membrane Proteins/metabolism , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Retina/embryology , Retina/growth & development , Retina/physiopathology , Reverse Transcriptase Polymerase Chain Reaction/methods
13.
Mol Vis ; 11: 657-64, 2005 Aug 30.
Article in English | MEDLINE | ID: mdl-16145543

ABSTRACT

PURPOSE: Mutations in ELOVL4, a member of the fatty acid elongase (ELO) family, are responsible for autosomal dominant Stargardt-like macular degeneration. The specific role of ELOVL4 in photoreceptors and the degenerative events induced by dominant ELOVL4 mutations are not well understood. As a first step to identifying possible mechanisms contributing to cellular dysfunction, we transfected HEK293 and COS cells with fluorescent-labeled wild-type and mutant ELOVL4 constructs. Effects of mutant ELOVL4 on interaction with wild-type protein were examined in this in vitro model. METHODS: Wild-type and mutant ELOVL4 proteins including ELOVL4 truncation (270X, a truncated ELOVL4 protein at amino acid position 270) and ELOVL4 5 bp deletion (5bp-del) and ELOVL4 (5A, substituting the ER retention signal, KAKGD, with a five alanine amino acid tract) were expressed as EGFP or DsRed fusion proteins. Cellular localization of these proteins was examined by fluorescence microscopy. ELOVL4 protein aggregates were measured by co-immunoprecipitation and by sucrose gradient centrifugation followed by immunodetection with western blots. To study cellular status of cells expressing mutant ELOVL4 proteins, transfected cells were examined for upregulation of Bip and CHOP, markers for the unfolded protein response (UPR) by western blotting. RESULTS: ELOVL4 mutants were not retained within the ER but were rather mislocalized and formed aggregates. Importantly, when cotransfected with wild-type ELOVL4, the mutants bound to and sequestered the wild-type protein into the aggregates. Expression of ELOVL4 mutants also induced UPR as evidenced by Bip and CHOP expression. CONCLUSIONS: Using this in vitro cell system, we have identified alterations in wild-type ELOVL4 protein localization, aggregate formation, and the induction of cellular stress by the ELOVL4 mutants. We propose that "inactivation" of the wild-type ELOVL4 protein through sequestration to a non-ER compartment by ELOVL4 mutants may play a role in cellular dysfunction.


Subject(s)
Endoplasmic Reticulum/metabolism , Eye Proteins/metabolism , Macular Degeneration/metabolism , Membrane Proteins/metabolism , Animals , Blotting, Western , COS Cells/metabolism , Centrifugation, Density Gradient , Chlorocebus aethiops , Endoplasmic Reticulum Chaperone BiP , Eye Proteins/genetics , Gene Expression , Heat-Shock Proteins/metabolism , Humans , Kidney/embryology , Kidney/metabolism , Macular Degeneration/genetics , Membrane Proteins/genetics , Microscopy, Fluorescence , Molecular Chaperones/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Transcription Factor CHOP/metabolism , Transfection , Up-Regulation
14.
Protein Expr Purif ; 40(1): 86-90, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15721775

ABSTRACT

Bacterial resistance to aminoglycosides continues to escalate and is widely recognized as a serious health threat, contributing to interest in understanding the mechanisms of resistance. One important mechanism of streptomycin modification is through ATP dependent O-adenylation, catalyzed by streptomycin adenylyltransferase (SMATase). The aim of this study was to purify the recombinant SMATase by Ni(2+)-IDA-His bind resin column chromatography. Thioredoxin-His6-tagged SMATase fusion protein was produced in a bacterial intracellular expression system mainly in a soluble form. The purified fusion protein showed a single band on SDS-PAGE corresponding to 49 kDa. The recovery of fusion protein was 47% with ninefold purification. The fusion system provided a single step, easy and very rapid purification of SMATase and is suitable for obtaining a highly purified functional protein of interest. The fusion does not affect the functionality of the protein.


Subject(s)
Escherichia coli/genetics , Nucleotidyltransferases/genetics , Nucleotidyltransferases/isolation & purification , Chromatography, Affinity , Nucleotidyltransferases/metabolism , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Thioredoxins/metabolism
15.
Hum Mol Genet ; 14(2): 255-65, 2005 Jan 15.
Article in English | MEDLINE | ID: mdl-15563508

ABSTRACT

Retina and retinal pigment epithelium (RPE) belong to the metabolically most active tissues in the human body. Efficient removal of acid load from retina and RPE is a critical function mediated by the choriocapillaris. However, the mechanism by which pH homeostasis is maintained is largely unknown. Here, we show that a functional complex of carbonic anhydrase 4 (CA4) and Na+/bicarbonate co-transporter 1 (NBC1) is specifically expressed in the choriocapillaris and that missense mutations in CA4 linked to autosomal dominant rod-cone dystrophy disrupt NBC1-mediated HCO3- transport. Our results identify a novel pathogenic pathway in which a defect in a functional complex involved in maintaining pH balances, but not expressed in retina or RPE, leads to photoreceptor degeneration. The importance of a functional CA4 for survival of photoreceptors implies that CA inhibitors, which are widely used as medications, particularly in the treatment of glaucoma, may have long-term adverse effects on vision.


Subject(s)
Carbonic Anhydrase IV/genetics , Mutation , Photoreceptor Cells/metabolism , Carbonic Anhydrase IV/metabolism , Female , Humans , Hydrogen-Ion Concentration , Male , Pedigree , Sodium-Bicarbonate Symporters/metabolism
16.
Invest Ophthalmol Vis Sci ; 45(12): 4263-7, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15557430

ABSTRACT

PURPOSE: To conduct clinical and genetic studies in a European family with autosomal dominant Stargardt-like macular dystrophy (adSTGD-like MD) and to investigate the functional consequences of a novel ELOVL4 mutation. METHODS: Ophthalmic examination and mutation screening by direct sequencing of the ELOVL4 gene was performed in two affected individuals. Wild-type and mutant ELOVL4 genes were expressed as enhanced green fluorescent protein (EGFP) fusion proteins in transient transfection in NIH-3T3 and HEK293 cells. To determine the subcellular localization of ELOVL4, an endoplasmic-reticulum (ER)-specific marker for pDsRed2-ER was cotransfected with ELOVL4 constructs. Transfected cells were viewed by confocal microscopy. Western blot analysis was performed to assess protein expression using an anti-GFP antibody. RESULTS: Affected patients exhibited macular atrophy with surrounding flecks characteristic of adSTGD-like MD. A novel ELOVL4 p.Tyr270X mutation was detected in affected individuals. In cell-transfection studies, wild-type ELOVL4 localized preferentially to the ER. In contrast, the mutant protein appeared to be mislocalized within transfected cells. CONCLUSIONS: In a European family with adSTGD-like MD, a novel ELOVL4 mutation was found to underlie the disorder. Transfection studies indicated that, unlike wild-type ELOVL4, the mutant protein does not localize to the ER but rather appears to be sequestered elsewhere in an aggregated pattern in the cytoplasm. Further analysis of the function of normal and mutant ELOVL4 will provide insight into the mechanism of macular degeneration.


Subject(s)
Eye Proteins/genetics , Genes, Dominant , Macular Degeneration/genetics , Membrane Proteins/genetics , Mutation , Adolescent , Adult , Atrophy , Blotting, Western , Cell Line , Codon, Terminator , Cytoplasm/metabolism , Endoplasmic Reticulum/metabolism , Eye Proteins/metabolism , Female , Green Fluorescent Proteins , Humans , Luminescent Proteins , Macula Lutea/pathology , Macular Degeneration/pathology , Macular Degeneration/physiopathology , Membrane Proteins/metabolism , Microscopy, Confocal , Pedigree , Tissue Distribution , Transfection , Tyrosine , Visual Acuity
17.
Ophthalmic Genet ; 25(2): 133-45, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15370544

ABSTRACT

Pattern dystrophy is a heterogeneous group of retinal dystrophies of which butterfly-shaped pattern dystrophy (BPD) and adult-onset foveomacular dystrophy (AOFMD) are the two most common forms. BPD is characterized by a butterfly-shaped, irregular, depigmented lesion at the level of the retinal pigment epithelium. In contrast, AOFMD is characterized by the presence of slightly elevated, symmetric, solitary, round to oval, yellow lesions at the level of the retinal pigment epithelium. We identified three independent kindreds with pattern dystrophy, one with four patients affected with BPD and the other two with 14 affected patients with AOFMD. We performed complete ophthalmic examination, fluorescein angiography, linkage mapping, and mutational screening in the RDS/peripherin gene in the affected patients. Patients affected with BPD had a best-corrected vision of 20/20 to 20/25, whereas vision in the eyes of patients with AOFMD ranged from 20/20 to 20/400. In all three kindreds, sequence analysis identified an A-to-G change at nucleotide position 422 of the RDS/peripherin gene, predicting a novel Tyr-141-Cys substitution. A haplotype analysis revealed that these three kindreds shared an identical disease haplotype at the RDS/peripherin locus, indicating that the mutation reflects a founder effect. The sequence change that segregated with the disease phenotype was not observed in 200 control chromosomes. Our results identified a novel mutation in the RDS/ peripherin gene that can cause diverse macular phenotypes. Genetic and clinical investigation of pattern dystrophy may provide useful diagnostic tools and new treatment strategies for this disorder.


Subject(s)
Intermediate Filament Proteins/genetics , Macular Degeneration/genetics , Macular Degeneration/pathology , Membrane Glycoproteins/genetics , Mutation/genetics , Nerve Tissue Proteins/genetics , Adult , Aged , Chromosome Mapping , DNA Mutational Analysis , Female , Fluorescein Angiography , Humans , Male , Middle Aged , Pedigree , Peripherins , Phenotype
18.
Mol Vis ; 10: 248-53, 2004 Mar 31.
Article in English | MEDLINE | ID: mdl-15073583

ABSTRACT

PURPOSE: ELOVL4 is a member of the fatty acid elongase (ELO) family of genes. Mutations of this gene are responsible for autosomal dominant Stargardt-like macular degeneration. However, the specific role of ELOVL4 in photoreceptor cells and the mechanism by which mutations in ELOVL4 causes macular degeneration are not known. In this study we examined the subcellular localization of wild type (wt) and mutant (mt) ELOVL4 EGFP fusion protein and the potential functional consequence of mtELOVL4 expression on cell viability. METHODS: Wt and mt ELOVL4 were expressed as EGFP fusion proteins in NIH 3T3 and HEK293 cells. Subcellular localizations of the fusion proteins were determined with a series of organelle-specific markers for endoplasmic reticulum (pDsRed2-ER), mitochondria (pDsRed2-Mito), peroxisomes (pDsRed2-Peroxi), and Golgi (BODIPY TR). Transfected cells were viewed using confocal and episcopic-fluorescence microscopy. Western blot analysis was performed to assess protein expression using an anti-GFP antibody. TUNEL staining was used to quantify apoptotic cell death. RESULTS: In cell transfection studies, wtELOVL4/EGFP fusion protein localized preferentially to the endoplasmic reticulum (ER) and was not found to be discernibly present in mitochondria, peroxisomes, or Golgi. In contrast, the truncated mutant fusion protein (which has no ER retention signal) appeared to be mislocalized to other compartments within transfected cells. Transfected cells expressing mtELOVL4/EGFP fusion protein exhibited induction of apoptotic cell death. CONCLUSIONS: Unlike wtELOVL4/EGFP fusion protein, the mtELOVL4/EGFP fusion protein did not localize to the ER but rather appeared to be sequestered elsewhere in an aggregated pattern in the cytoplasm. The apoptosis induced by the mutant ELOVL4 fusion protein may be the mechanism whereby photoreceptor cells degenerate in Stargardt-like macular degeneration. Our study has provided an important in vitro model system for further assessment of ELOVL4 biochemical functions.


Subject(s)
Apoptosis , Eye Proteins/metabolism , Membrane Proteins/metabolism , Animals , Blotting, Western , Cell Survival , Cytoplasm/metabolism , Endoplasmic Reticulum/metabolism , Eye Proteins/genetics , Green Fluorescent Proteins , Humans , Immunoblotting , Kidney/cytology , Kidney/embryology , Kidney/metabolism , Luminescent Proteins/metabolism , Membrane Proteins/genetics , Mice , Microscopy, Confocal , Microscopy, Fluorescence , Mutation , NIH 3T3 Cells/metabolism , Recombinant Fusion Proteins/metabolism , Subcellular Fractions , Transfection
19.
Mol Vis ; 9: 301-7, 2003 Jul 03.
Article in English | MEDLINE | ID: mdl-12847421

ABSTRACT

PURPOSE: Stargardt-like macular dystrophy (STGD3) is an autosomal dominant form of early onset macular degeneration. The disease causing gene ELOVL4 encodes a protein that belongs to a family of proteins functioning in elongation of long chain fatty acids. The purpose of this study is to characterize cross-species conservation of ELOVL4 and investigate its mRNA distribution in the developing mouse eye. METHODS: Bovine and porcine orthologs of the human ELOVL4 gene were cloned using RT-PCR method. EST and HTGS databases were searched for orthologs of ELOVL4. Cross-species alignments were performed using ClustalW. In situ hybridizations using murine Elovl4 probes were performed on frozen sections of mouse eyes. RESULTS: Elovl4 orthologs from mammalian to invertebrate species share strong sequence homology with human ELOVL4 at the amino acid level, suggesting functional conservation of Elovl4 during evolution. Expression of Elovl4 in mouse retina begins at E15 during embryogenesis and persists in postnatal stages. However, Elovl4 is predominantly expressed in the retinal ganglion cells at P1-P3, followed by predominant expression in the outer nuclear layer at P7, with its final expression enriched in inner segments of photoreceptors. CONCLUSIONS: Elovl4 expression in developing retina follows a dynamic pattern. It switches from predominant ganglion cell expression in embryonic and early postnatal development to predominant expression in the photoreceptor inner segments in later stages. Phylogenetic analysis reveals strong conservation of Elovl4 among different species throughout the vertebrate subphylum consistent with our hypothesis that ELOVL4 performs a fundamentally important function.


Subject(s)
Eye Proteins/genetics , Membrane Proteins/genetics , RNA, Messenger/metabolism , Retina/embryology , Amino Acid Sequence , Animals , Cattle , Cloning, Molecular , Humans , In Situ Hybridization , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Phylogeny , Retina/growth & development , Retina/metabolism , Retinal Ganglion Cells/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Swine
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