Isoforms of SLC26A6 mediate anion transport and have functional PDZ interaction domains

2003 ◽  
Vol 284 (3) ◽  
pp. C769-C779 ◽  
Author(s):  
Hannes Lohi ◽  
Georg Lamprecht ◽  
Daniel Markovich ◽  
Anders Heil ◽  
Minna Kujala ◽  
...  
Keyword(s):  
Splice Variants ◽  
Functional Characterization ◽  
Regulatory Protein ◽  
Functional Expression ◽  
General Concept ◽  
Specific Gene ◽  
Anion Transporters ◽  
A Genome ◽  
Alternatively Spliced

The solute carrier gene family SLC26 consists of tissue-specific anion exchanger genes, three of them associated with distinct human recessive disorders. By a genome-driven approach, several new SLC26 family members have been identified, including a kidney- and pancreas-specific gene, SLC26A6. We report the functional characterization of SLC26A6 and two new alternatively spliced variants, named SLC26A6c and SLC26A6d. Immunofluorescence studies on transiently transfected cells indicated membrane localization and indicated that both NH2- and COOH-terminal tails of the SLC26A6 variants are located intracellularly, suggesting a topology with an even number of transmembrane domains. Functional expression of the three proteins in Xenopus oocytes demonstrated Cl− and SO[Formula: see text] transport activity. In addition, the transport of SO[Formula: see text] and Cl− was inhibited by DIDS and HCO[Formula: see text]. We demonstrated also that the COOH terminus of SLC26A6 binds to the first and second PDZ domains of the Na+/H+ exchanger (NHE)3 kinase A regulatory protein (E3KARP) and NHE3 regulatory factor (NHERF) proteins in vitro. Truncation of the last three amino acids (TRL) of SLC26A6 abrogated the interaction but did not affect transport function. These results demonstrate that SLC26A6 and its two splice variants can function as anion transporters linked to PDZ-interaction pathways. Our results support the general concept of microdomain organization for ion transport and suggest a mechanism for cystic fibrosis transmembrane regulator (CFTR)-mediated SLC26A6 upregulation in pancreatic duct cells.

scholarly journals Congenital Lipoid Adrenal Hyperplasia: Functional Characterization of Three Novel Mutations in the STAR Gene

2010 ◽  
Vol 95 (3) ◽  
pp. 1301-1308 ◽  
Author(s):  
Susanne Bens ◽  
Angelika Mohn ◽  
Bilgin Yüksel ◽  
Alexandra E. Kulle ◽  
Matthias Michalek ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Regulatory Protein ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Adrenal Hyperplasia ◽  
Turkish Patient ◽  
Star Protein ◽  
Sex Development ◽  
Congenital Lipoid Adrenal Hyperplasia

Abstract Context: The steroidogenic acute regulatory protein (StAR) has been shown to be essential for steroidogenesis by mediating cholesterol transfer into mitochondria. Inactivating StAR mutations cause the typical clinical picture of congenital lipoid adrenal hyperplasia. Objective: The objective of the investigation was to study the functional and structural consequences of three novel StAR mutations (p.N148K in an Italian patient; p.P129fs and p.Q128R in a Turkish patient). Methods and Results: Transient in vitro expression of the mutant proteins together with P450 side-chain cleavage enzyme, adrenodoxin, and adrenodoxin reductase yielded severely diminished cholesterol conversion of the p.N148K mutant, the combined p.P129fs and p.Q128R mutant, and the p.P129fs mutant by itself. The p.Q128R mutant led to a higher cholesterol conversion than the wild-type StAR protein. As derived from three-dimensional protein modeling, the residue N148 is lining the ligand cavity of StAR. A positively charged lysine residue at position 148 disturbs the hydrophobic cluster formed by the α4-helix and the sterol binding pocket. The frame shift mutation p.P129fs truncates the StAR protein. Residue p.Q128 is situated at the surface of the molecule and is not part of any functionally characterized region of the protein. Conclusion: The mutations p.N148K and p.P129fs cause adrenal insufficiency in both cases and lead to a disorder of sex development with complete sex reversal in the 46, XY case. The mutation p.Q128R, which is not relevant for the patient’s phenotype, is the first reported variant showing a gain of function. We speculate that the substitution of hydrophilic glutamine with basic arginine at the surface of the molecule may accelerate cholesterol transfer.

scholarly journals Structural analysis of the DNA-binding domain of alternatively spliced steroid receptors

2002 ◽  
Vol 173 (3) ◽  
pp. 429-436 ◽  
Author(s):  
L Wickert ◽  
J Selbig
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Splice Variants ◽  
Steroid Receptor ◽  
Binding Domain ◽  
Dna Binding Domain ◽  
Amino Acid Residues ◽  
Receptor Function ◽  
Alternatively Spliced

We have generated 24 DNA-binding domain structure models of alternatively spliced or mutated steroid receptor variants by homology-based modeling. Members of the steroid receptor family dispose of a DNA-binding domain which is built from two zinc fingers with a preserved sequence homology of about 90%. Data from crystallographic analysis of the glucocorticoid receptor DNA-binding domain are therefore appropriate to serve as a template structure. We inserted or deleted amino acid residues in order to study the structural details of the glucocorticoid, mineralocorticoid, and androgen receptor splice variants. The receptor variants are created by QUANTA- and MODELLER-based modeling. Subsequently, the resulting energy-minimized models were compared with each other and with the wild-type receptor respectively. A prediction for the receptor function based mainly on the preservation or destruction of secondary structures has been carried out. The simulations showed that amino acid insertions of one, four or nine additional residues of existing steroid receptor splice variants were tolerated without destruction of the secondary structure. In contrast, a deletion of four amino acids at the splice site junction leads to modifications in the secondary structure of the DNA-recognition helix which apparently disturb the receptor-DNA interaction. Furthermore, an insertion of 23 amino acid residues between the zinc finger of the androgen receptor leads to a large loop with an additional alpha-helical structure which seems to disconnect a specific contact from its hormone response element. Thereafter, the prediction of receptor function based on the molecular models was compared with the available experimental results from the in vitro function tests. We obtained a close correspondence between the molecular modeling-based predictions and the conclusions of receptor function drawn from in vitro studies.

scholarly journals Dysregulated Alternative Splicing Pattern of PKCδ during Differentiation of Human Preadipocytes Represents Distinct Differences between Lean and Obese Adipocytes

ISRN Obesity ◽  
2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Gay Carter ◽  
André Apostolatos ◽  
Rekha Patel ◽  
Abhishek Mathur ◽  
Denise Cooper ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Expression Pattern ◽  
Molecular Mechanisms ◽  
Developmental Process ◽  
Splice Variants ◽  
Apoptosis Pathway ◽  
Antiapoptotic Proteins ◽  
Alternatively Spliced ◽  
Human Preadipocytes

Obesity and its comorbidities affect millions of people. Here, we demonstrate that human preadipocytes are susceptible to programmed cell death (apoptosis) while mature adipocytes are resistant to apoptosis. The molecular mechanisms underlying the phenotype of apoptosis-resistant adipocytes are lesser known. To study the role of apoptosis and define molecular differences in the developmental process of adipogenesis, human preadipocytes were differentiated in vitro to mature adipocytes. Many genes in the apoptosis pathway are alternatively spliced. Our data demonstrates that during differentiation PKCδ, Bclx, and Caspase9 switch to their prosurvival splice variants along with an increase in Bcl2 expression when the cells terminally differentiate into mature adipocytes. Next we determined the expression pattern of these genes in obesity. Our data indicated high expression of PKCδVIII in adipose tissue of obese patient in different depots. We demonstrate a shift in the in vitro expression of these splice variants in differentiating preadipocytes derived from obese patients along with a decrease in adipogenesis markers. Hence, the programmed splicing of antiapoptotic proteins is a pivotal switch in differentiation that commits adipocytes to a prosurvival pathway. The expression pattern of these genes is dysregulated in obesity and may contribute to adipose tissue dysfunction.

scholarly journals Clathrin light chain diversity regulates membrane deformation in vitro and synaptic vesicle formation in vivo

2020 ◽  
Vol 117 (38) ◽  
pp. 23527-23538 ◽  
Author(s):  
Lisa Redlingshöfer ◽  
Faye McLeod ◽  
Yu Chen ◽  
Marine D. Camus ◽  
Jemima J. Burden ◽  
...  
Keyword(s):  
Synaptic Vesicle ◽  
Light Chain ◽  
Splice Variants ◽  
Membrane Deformation ◽  
Functional Balance ◽  
Electrophysiological Recordings ◽  
Alternatively Spliced ◽  
Vesicle Pool

Clathrin light chain (CLC) subunits in vertebrates are encoded by paralogous genesCLTAandCLTB, and both gene products are alternatively spliced in neurons. To understand how this CLC diversity influences neuronal clathrin function, we characterized the biophysical properties of clathrin comprising individual CLC variants for correlation with neuronal phenotypes of mice lacking either CLC-encoding gene. CLC splice variants differentially influenced clathrin knee conformation within assemblies, and clathrin with neuronal CLC mixtures was more effective in membrane deformation than clathrin with single neuronal isoforms nCLCa or nCLCb. Correspondingly, electrophysiological recordings revealed that neurons from mice lacking nCLCa or nCLCb were both defective in synaptic vesicle replenishment. Mice with only nCLCb had a reduced synaptic vesicle pool and impaired neurotransmission compared to WT mice, while nCLCa-only mice had increased synaptic vesicle numbers, restoring normal neurotransmission. These findings highlight differences between the CLC isoforms and show that isoform mixing influences tissue-specific clathrin activity in neurons, which requires their functional balance.

scholarly journals A unique spliced varicella-zoster virus latency transcript represses expression of the viral transactivator gene 61

2017 ◽  
Author(s):  
Daniel P. Depledge ◽  
Werner J. D. Ouwendijk ◽  
Tomohiko Sadaoka ◽  
Shirley E. Braspenning ◽  
Yasuko Mori ◽  
...  
Keyword(s):  
Varicella Zoster Virus ◽  
Functional Characterization ◽  
Skin Lesions ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Viral Transactivator ◽  
Virus Latency ◽  
Latently Infected ◽  
Alternatively Spliced

During primary infection, neurotropic alphaherpesviruses (αHVs) gain access to neurons in sensory and cranial ganglia establishing lifelong latent infection from which they can later reactivate to cause debilitating disease1. For most αHVs, including the best-studied herpes simplex type 1 ( HSV-1), viral latency is characterized by expression of a single or restricted set of transcripts that map antisense to the open reading frame (ORF) homologous to the major HSV immediate early viral transactivator, ICP02. These latency transcripts, either directly or through encoded miRNAs or proteins, repress expression of the ICP0 orthologues3–5. The exception is varicella-zoster virus (VZV), an αHV which infects over 90% of adults and for which neither a canonical latency transcript1,6–8 nor a putative mechanism for repressing lytic transcription during latency have been identified. Here, we describe the discovery and functional characterization of a VZV latency transcript (VLT), that maps antisense to VZV ORF 61 (the VZV ICP0 homologue9,10), and which is consistently expressed in neurons of latently infected human trigeminal ganglia (TG). VLT encodes a protein with late kinetics during lytic VZV infection in vitro and in zoster skin lesions. Whereas multiple alternatively spliced VLT isoforms are expressed during lytic VZV infection, a single unique VLT isoform that specifically suppresses ORF61 gene expression predominates in latently VZV-infected human TG. The discovery of VLT directly unifies the latent VZV transcription program with those of better-characterized αHVs, removing longstanding barriers to understanding VZV latency and paving the way for research into the development of vaccines that do not establish latency or reactivate, and drugs that eradicate latent VZV.

Prokaryotic functional expression and activity comparison of three CYP9A genes from the polyphagous pestHelicoverpa armigera

2017 ◽  
Vol 108 (1) ◽  
pp. 77-83 ◽  
Author(s):  
D. Liu ◽  
K. Tian ◽  
Y. Yuan ◽  
M. Li ◽  
M. Zheng ◽  
...  
Keyword(s):  
Pyrethroid Resistance ◽  
Functional Characterization ◽  
Kinetic Studies ◽  
Functional Expression ◽  
Cytochrome P450 Reductase ◽  
E Coli ◽  
Important Pest ◽  
Activity Comparison

AbstractCytochrome P450s (CYPs or P450s) have been long recognized as very important enzymes in the metabolism of xenobiotic and endogenous compounds, but only a few CYPs have been functionally characterized in insects. The effort in functional characterization of insect P450s is heavily hindered by technical difficulties in preparing active, individual P450 enzymes directly from the target insect. In this paper, we describe the functional expression of two additional pyrethroid resistance-associated CYP9A genes (CYP9A12andCYP9A17) from the polyphagous pestHelicoverpa armigerain the facileEscherichia coli. The functionality ofE. coliproduced CYP9A12, CYP9A14, and CYP9A17 was investigated and activities of these CYP9As were compared against three probe substrates after reconstitution with NADPH-dependent cytochrome P450 reductase. The results showed that active forms of CYP9A12 and CYP9A17 were expressed inE. coliwith a content of about 1.0–1.5 nmol mg−1protein in membrane preparations.In vitroassays showed that CYP9A14 was capable of catalyzing O-dealkylation of methoxyresorufin (MROD), ethoxyresorufin (EROD), and benzyloxyresorufin (BROD), while CYP9A12 and CYP9A17 exhibited only MROD and EROD activities. Kinetic studies demonstrated that CYP9A14 had the greatestkcat/Kmvalue for MROD, and CYP9A17 for EROD, while the lowestkcat/Kmvalues for both MROD and EROD were observed for CYP9A12. The distinct biochemical traits suggest that the three paralogous CYP9As may play different roles in xenobiotic metabolism in this important pest.

scholarly journals Identification and Functional Characterization of Neo-Poly(A) Polymerase, an RNA Processing Enzyme Overexpressed in Human Tumors

2001 ◽  
Vol 21 (16) ◽  
pp. 5614-5623 ◽  
Author(s):  
Suzanne L. Topalian ◽  
Syuzo Kaneko ◽  
Monica I. Gonzales ◽  
Gareth L. Bond ◽  
Yvona Ward ◽  
...  
Keyword(s):  
Rna Processing ◽  
Blot Analysis ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Human Cancer ◽  
Functional Characterization ◽  
Sequence Divergence ◽  
In Vitro Assays ◽  
Processing Enzyme

ABSTRACT Poly(A) polymerase (PAP) plays an essential role in polyadenylation of mRNA precursors, and it has long been thought that mammalian cells contain only a single PAP gene. We describe here the unexpected existence of a human PAP, which we call neo-PAP, encoded by a previously uncharacterized gene. cDNA was isolated from a tumor-derived cDNA library encoding an 82.8-kDa protein bearing 71% overall similarity to human PAP. Strikingly, the organization of the two PAP genes is nearly identical, indicating that they arose from a common ancestor. Neo-PAP and PAP were indistinguishable in in vitro assays of both specific and nonspecific polyadenylation and also endonucleolytic cleavage. Neo-PAP produced by transfection was exclusively nuclear, as demonstrated by immunofluorescence microscopy. However, notable sequence divergence between the C-terminal domains of neo-PAP and PAP suggested that the two enzymes might be differentially regulated. While PAP is phosphorylated throughout the cell cycle and hyperphosphorylated during M phase, neo-PAP did not show evidence of phosphorylation on Western blot analysis, which was unexpected in the context of a conserved cyclin recognition motif and multiple potential cyclin-dependent kinase (cdk) phosphorylation sites. Intriguingly, Northern blot analysis demonstrated that each PAP displayed distinct mRNA splice variants, and both PAP mRNAs were significantly overexpressed in human cancer cells compared to expression in normal or virally transformed cells. Neo-PAP may therefore be an important RNA processing enzyme that is regulated by a mechanism distinct from that utilized by PAP.

Insights into the biochemical and functional characterization of sortase E transpeptidase of Corynebacterium glutamicum

2019 ◽  
Vol 476 (24) ◽  
pp. 3835-3847 ◽  
Author(s):  
Aliyath Susmitha ◽  
Kesavan Madhavan Nampoothiri ◽  
Harsha Bajaj
Keyword(s):  
Protein Engineering ◽  
Cell Attachment ◽  
Functional Characterization ◽  
Protein Structures ◽  
Structural Similarity ◽  
Surface Proteins ◽  
Sortase A ◽  
Peptide Cleavage ◽  
New Findings

Most Gram-positive bacteria contain a membrane-bound transpeptidase known as sortase which covalently incorporates the surface proteins on to the cell wall. The sortase-displayed protein structures are involved in cell attachment, nutrient uptake and aerial hyphae formation. Among the six classes of sortase (A–F), sortase A of S. aureus is the well-characterized housekeeping enzyme considered as an ideal drug target and a valuable biochemical reagent for protein engineering. Similar to SrtA, class E sortase in GC rich bacteria plays a housekeeping role which is not studied extensively. However, C. glutamicum ATCC 13032, an industrially important organism known for amino acid production, carries a single putative sortase (NCgl2838) gene but neither in vitro peptide cleavage activity nor biochemical characterizations have been investigated. Here, we identified that the gene is having a sortase activity and analyzed its structural similarity with Cd-SrtF. The purified enzyme showed a greater affinity toward LAXTG substrate with a calculated KM of 12 ± 1 µM, one of the highest affinities reported for this class of enzyme. Moreover, site-directed mutation studies were carried to ascertain the structure functional relationship of Cg-SrtE and all these are new findings which will enable us to perceive exciting protein engineering applications with this class of enzyme from a non-pathogenic microbe.

Neuroprotective and anti-amnestic effects of a combination therapy in a model of photochemical ischemic damage in the prefrontal cortex

2018 ◽  
pp. 39-45
Author(s):  
Ф.М. Шакова ◽  
Т.И. Калинина ◽  
М.В. Гуляев ◽  
Г.А. Романова
Keyword(s):  
Prefrontal Cortex ◽  
Nerve Growth Factor ◽  
Growth Factor ◽  
Combination Therapy ◽  
Neuroprotective Effect ◽  
Regulatory Protein ◽  
Human Growth ◽  
Neuroprotective Effects ◽  
Nerve Growth

Цель исследования - изучение влияния комбинированной терапии (мутантные молекулы эритропоэтина (EPO) и дипептидный миметик фактора роста нервов ГК-2H) на воспроизведение условного рефлекса пассивного избегания (УРПИ) и объем поражения коры мозга у крыс с двусторонним ишемическим повреждением префронтальной коры. Методика. Мутантные молекулы EPO (MЕРО-TR и MЕPО-Fc) с значительно редуцированной эритропоэтической и выраженной цитопротекторной активностью созданы методом генной инженерии. Используемый миметик фактора роста нервов человека, эндогенного регуляторного белка, в экспериментах in vitro проявлял отчетливые нейропротективные свойства. Двустороннюю фокальную ишемию префронтальной коры головного мозга крыс создавали методом фотохимического тромбоза. Выработку и оценку УРПИ проводили по стандартной методике. Объем повреждения мозга оценивался при помощи МРТ. MEPO-TR и MEPO-Fc (50 мкг/кг) вводили интраназально однократно через 1 ч после фототромбоза, ГК-2Н (1 мг/кг) - внутрибрюшинно через 4 ч после фототромбоза и далее в течение 4 послеоперационных суток. Результаты. Выявлено статистически значимое сохранение выработанного до ишемии УРПИ, а также значимое снижение объема повреждения коры при комплексной терапии. Полученные данные свидетельствуют об антиамнестическом и нейропротекторном эффектах примененной комбинированной терапии, которые наиболее отчетливо выражены в дозах: МEPO-Fc (50 мкг/кг) и ГК-2Н (1 мг/кг). Заключение. Подтвержден нейропротекторный эффект и усиление антиамнестического эффекта при сочетанном применении мутантных производных эритропоэтина - MEPO-TR и MEPO-Fc и дипептидного миметика фактора роста нервов человека ГК-2H. The aim of this study was to investigate the effect of combination therapy, including mutant erythropoietin molecules (EPO) and a dipeptide mimetic of the nerve growth factor, GK-2H, on the conditioned passive avoidance (PA) reflex and the volume of injury induced by bilateral ischemia of the prefrontal cortex in rats. Using the method of genetic engineering the mutant molecules of EPO, MERO-TR and MEPO-Fc, with strongly reduced erythropoietic and pronounced cytoprotective activity were created. The used human nerve growth factor mimetic, an endogenous regulatory protein based on the b-bend of loop 4, which is a dimeric substituted dipeptide of bis- (N-monosuccinyl-glycyl-lysine) hexamethylenediamine, GK-2 human (GK-2H), has proven neuroprotective in in vitro experiments. Methods. Bilateral focal ischemic infarction was modeled in the rat prefrontal cortex by photochemically induced thrombosis. The PA test was performed according to a standard method. Volume of brain injury was estimated using MRI. MEPO-TR, and MEPO-Fc (50 mg/kg, intranasally) were administered once, one hour after the injury. GK-2Н (1 mg/kg, i.p.) was injected four hours after the injury and then for next four days. Results. The study showed that the complex therapy provided statistically significant retention of the PA reflex developed prior to ischemia and a significant decrease in the volume of injury. The anti-amnestic and neuroprotective effects of combination therapy were most pronounced at doses of MEPO-Fc 50 mg/kg and GK-2H 1 mg/kg. Conclusion. This study has confirmed the neuroprotective effect and enhancement of the anti-amnestic effect exerted by the combination of mutant erythropoietin derivatives, MEPO-TR and MEPO-Fc, and the dipeptide mimetic of human growth factor GK-2H.

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