Subtype identification and functional characterization of ryanodine receptors in rat cerebral artery myocytes

Ryanodine receptors (RyRs) regulate contractility in resistance-size cerebral artery smooth muscle, yet their molecular identity, subcellular location, and phenotype in this tissue remain unknown. Following rat resistance-size cerebral artery myocyte sarcoplasmic reticulum (SR) purification and incorporation into POPE-POPS-POPC (5:3:2; wt/wt) bilayers, unitary conductances of 110 ± 8, 334 ± 15, and 441 ± 27 pS in symmetric 300 mM Cs+ were usually detected. The most frequent (34/40 bilayers) conductance (334 pS) decreased to ≤100 pS when Cs+ was replaced with Ca2+. The predominant conductance displayed 66 bursts/min with at least three open and three closed states. The steady-state activity (NPo)-voltage curve was bell shaped, with NPo drastically decreasing when voltage was switched from −30 to −40 mV. NPo increased when intracellular calcium (Ca2+i) was raised within 0.1–100 μM to abruptly diminish with higher Ca2+i. Thus maximal activity occurred within the Ca2+i range found in rat cerebral artery myocytes under physiological conditions. NPo was reduced by ruthenium red (80 μM), increased monotonically by caffeine (0.1–5 mM) or ryanodine (0.05–5 μM), and unaffected by heparin (2 mg/ml). This phenotype resembles that of cardiac RyR and recombinant RyR2. RT-PCR detected RyR1, RyR2, and RyR3 transcripts in cerebral artery myocytes. However, real-time PCR indicated that RyR2 was 4 and 1.5 times more abundant than RyR1 and RyR3, respectively. Consistently, Western blotting showed that the RyR2 product was very abundant. Immunofluorescence showed that each RyR isoform distributed differentially among subcellular compartments. In particular, RyR2 was drastically stronger in the subplasmalemma than in other compartments, underscoring the predominance of RyR2 in a compartment where SR is abundant. Consistently, RyR from SR-enriched membranes displayed pharmacological specificity typical of RyR2, being activated by digoxin (1 μM), resistant to dantrolene (100 μM), and shifted to a subconductance by neomycin (100 nM). Therefore, RyR2 is the predominant molecular and functional RyR that is expressed in the SR membrane of rat resistance-size cerebral artery myocytes.

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Novel biochemical and functional insights into nuclear Ca2+ transport through IP3Rs and RyRs in osteoblasts

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.5.f784 ◽

2000 ◽

Vol 278 (5) ◽

pp. F784-F791 ◽

Cited By ~ 21

Author(s):

Olugbenga A. Adebanjo ◽

Gopa Biswas ◽

Baljit S. Moonga ◽

Hindupur K. Anandatheerthavarada ◽

Li Sun ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Ryanodine Receptors ◽

Functional Characterization ◽

Inositol Trisphosphate Receptors ◽

Nuclear Membranes ◽

Sds Page ◽

Direct Regulation

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab34) and anti-IP3R (Ab40) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab40, anti-RyR-1, anti-RyR-2 (Ab129), and anti-RyR-3 (Ab180). Only anti-RyR-1 and Ab40 showed bands corresponding, respectively, to full-length RyR-1 (∼500 kDa) and IP3R-1 (∼250 kDa). Band intensity was reduced by just ∼20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+ concentration ([Ca2+]np) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2+ via Ca2+-ATPase activation (1 mM ATP and ∼100 nM Ca2+). Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3 or cADP ribose resulted in a rapid and sustained [Ca2+]np elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+ influx in osteoblasts through nuclear membrane-resident IP3Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+pathway.

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Leaping into the Unknown World of Sporisorium scitamineum Candidate Effectors

Journal of Fungi ◽

10.3390/jof6040339 ◽

2020 ◽

Vol 6 (4) ◽

pp. 339

Author(s):

Natália Sousa Teixeira-Silva ◽

Patrícia Dayane Carvalho Schaker ◽

Hugo Vianna Silva Rody ◽

Thiago Maia ◽

Christopher M. Garner ◽

...

Keyword(s):

Plant Cell Wall ◽

Expression Profiles ◽

Functional Characterization ◽

Subcellular Location ◽

Sporisorium Scitamineum ◽

Effector Genes ◽

Molecular Events ◽

Phosphatase 2A ◽

Set Up

Sporisorium scitamineum is a biotrophic fungus causing sugarcane smut disease. In this study, we set up a pipeline and used genomic and dual transcriptomic data previously obtained by our group to identify candidate effectors of S. scitamineum and their expression profiles in infected smut-resistant and susceptible sugarcane plants. The expression profile of different genes after infection in contrasting sugarcane genotypes assessed by RT-qPCR depended on the plant genotypes and disease progression. Three candidate effector genes expressed earlier only in resistant plants, four expressed in both genotypes, and three later in susceptible plants. Ten genes were cloned and transiently expressed in N. benthamiana leaves to determine their subcellular location, while four localized in more than one compartment. Two candidates, g3890 having a nucleoplasmic and mitochondrial location and g5159 targeting the plant cell wall, were selected to obtain their possible corresponding host targets using co-immunoprecipitation (CoIP) experiments and mass spectrometry. Various potential interactors were identified, including subunits of the protein phosphatase 2A and an endochitinase. We investigated the presence of orthologs in sugarcane and using transcriptome data present their expression profiles. Orthologs of sugarcane shared around 70% similarity. Identifying a set of putative fungal effectors and their plant targets provides a valuable resource for functional characterization of the molecular events leading to smut resistance in sugarcane plants and uncovers further opportunities for investigation.

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Functional characterization of polypeptide release factor 1b in the ciliate Euplotes

Bioscience Reports ◽

10.1042/bsr20090154 ◽

2010 ◽

Vol 30 (6) ◽

pp. 425-431 ◽

Cited By ~ 9

Author(s):

Yan Wang ◽

Baofeng Chai ◽

Wei Wang ◽

Aihua Liang

Keyword(s):

Stop Codon ◽

Functional Characterization ◽

Subcellular Location ◽

Class I ◽

Release Factor ◽

Amino Acid Motif ◽

Codon Recognition ◽

Stop Codon Recognition ◽

Higher Eukaryotes

In higher eukaryotes, RF-I (class I release factor) [eRF1 (eukaryotic release factor 1)] is responsible for stop codon recognition and promotes nascent polypeptide release from the ribosome. Interestingly, two class I RFs, eRF1a and eRF1b, have been identified among the ciliates Euplotes, which are variant code organisms. In the present study, we analysed the comparative expression of eRF1a and eRF1b in Euplotes cells, demonstrating that the expression of eRF1b was higher than that of eRF1a. An interaction between eRF1b and eRF3 was confirmed, suggesting that an eRF1b function is facilitated by eRF3. Co-localization of both eRF1s indicated that they function in the same subcellular location in Euplotes cells. We also analysed the characteristics of stop codon discrimination by eRF1b. Like eRF1a, eRF1b recognized UAA and UAG as stop codons, but not UGA. This finding disagreed with the deduced characteristics of eRF1a/eRF1b from the classic hypothesis of ‘anticodon-mimicry’ proposed by Muramatsu et al. [Muramatsu, Heckmann, Kitanaka and Kuchino (2001) FEBS Lett. 488, 105–109]. Mutagenesis experiments indicated that the absolutely conserved amino acid motif ‘G31T32’ (numbered as for human eRF1) in eRF1b was the key to efficient stop codon recognition by eRF1b. In conclusion, these findings support and improve the ‘cavity model’ of stop codon discrimination by eRF1 proposed by Bertram et al. [Bertram, Bell, Ritchie, Fullerton and Stansfield (2000) RNA 6, 1236–1247] and Inagaki et al. [Inagaki, Blouin, Doolittle and Roger (2002) Nucleic Acids Res. 30, 532–544].

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Niemann-Pick type C disease: Subcellular location and functional characterization of NPC2 proteins with naturally occurring missense mutations

Human Mutation ◽

10.1002/humu.20173 ◽

2005 ◽

Vol 26 (1) ◽

pp. 20-28 ◽

Cited By ~ 17

Author(s):

Karim Chikh ◽

Céline Rodriguez ◽

Sébastien Vey ◽

Marie T. Vanier ◽

Gilles Millat

Keyword(s):

Functional Characterization ◽

Subcellular Location ◽

Missense Mutations ◽

Naturally Occurring ◽

Type C ◽

Niemann Pick

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Functional Characterization of a New GH107 Endo-α-(1,4)-Fucoidanase from the Marine Bacterium Formosa haliotis

Marine Drugs ◽

10.3390/md18110562 ◽

2020 ◽

Vol 18 (11) ◽

pp. 562

Author(s):

Marlene Vuillemin ◽

Artem S. Silchenko ◽

Hang Thi Thuy Cao ◽

Maxim S. Kokoulin ◽

Vo Thi Dieu Trang ◽

...

Keyword(s):

Marine Bacterium ◽

Divalent Cations ◽

Recombinant Expression ◽

Biological Activities ◽

Functional Characterization ◽

Maximal Activity ◽

Brown Macroalgae ◽

Pharmaceutical Uses ◽

Enzymatic Depolymerization

Fucoidans from brown macroalgae are sulfated fucose-rich polysaccharides, that have several beneficial biological activities, including anti-inflammatory and anti-tumor effects. Controlled enzymatic depolymerization of the fucoidan backbone can help produce homogeneous, defined fucoidan products for structure-function research and pharmaceutical uses. However, only a few endo-fucoidanases have been described. This article reports the genome-based discovery, recombinant expression in Escherichia coli, stabilization, and functional characterization of a new bacterial endo-α-(1,4)-fucoidanase, Fhf1, from Formosa haliotis. Fhf1 catalyzes the cleavage of α-(1,4)-glycosidic linkages in fucoidans built of alternating α-(1,3)-/α-(1,4)-linked l-fucopyranosyl sulfated at C2. The native Fhf1 is 1120 amino acids long and belongs to glycoside hydrolase (GH) family 107. Deletion of the signal peptide and a 470 amino acid long C-terminal stretch led to the recombinant expression of a robust, minimized enzyme, Fhf1Δ470 (71 kDa). Fhf1Δ470 has optimal activity at pH 8, 37–40 °C, can tolerate up to 500 mM NaCl, and requires the presence of divalent cations, either Ca2+, Mn2+, Zn2+ or Ni2+, for maximal activity. This new enzyme has the potential to serve the need for controlled enzymatic fucoidan depolymerization to produce bioactive sulfated fucoidan oligomers.

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Detection and functional characterization of ryanodine receptors from sea urchin eggs

The Journal of Physiology ◽

10.1111/j.1469-7793.1998.155bz.x ◽

1998 ◽

Vol 510 (1) ◽

pp. 155-164 ◽

Cited By ~ 27

Author(s):

Andrew J. Lokuta ◽

Alberto Darszon ◽

Carmen Beltrán ◽

Hector H. Valdivia

Keyword(s):

Sea Urchin ◽

Ryanodine Receptors ◽

Functional Characterization ◽

Sea Urchin Eggs

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Characterization of a UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase with an unusual lectin domain from the platyhelminth parasite Echinococcus granulosus

Biochemical Journal ◽

10.1042/bj20031877 ◽

2004 ◽

Vol 382 (2) ◽

pp. 501-510 ◽

Cited By ~ 20

Author(s):

Teresa FREIRE ◽

Cecilia FERNÁNDEZ ◽

Cora CHALAR ◽

Rick M. MAIZELS ◽

Pedro ALZARI ◽

...

Keyword(s):

Echinococcus Granulosus ◽

Transmembrane Domain ◽

Functional Characterization ◽

Maximal Activity ◽

Structural Features ◽

Helminth Parasites ◽

Lectin Domain ◽

C Terminus ◽

The One

As part of a general project aimed at elucidating the initiation of mucin-type O-glycosylation in helminth parasites, we have characterized a novel ppGalNAc-T (UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase) from the cestode Echinococcus granulosus (Eg-ppGalNAc-T1). A full-length cDNA was isolated from a library of the tissue-dwelling larval stage of the parasite, and found to code for a 654-amino-acid protein containing all the structural features of ppGalNAc-Ts. Functional characterization of a recombinant protein lacking the transmembrane domain showed maximal activity at 28 °C, in the range 6.5–7.5 pH units and in the presence of Cu2+. In addition, it transferred GalNAc to a broad range of substrate peptides, derived from human mucins and O-glycosylated parasite proteins, including acceptors containing only serine or only threonine residues. Interestingly, the C-terminal region of Eg-ppGalNAc-T1 bears a highly unusual lectin domain, considerably longer than the one from other members of the family, and including only one of the three ricin B repeats generally present in ppGalNAc-Ts. Furthermore, a search for conserved domains within the protein C-terminus identified a fragment showing similarity to a recently defined domain, specialized in the binding of organic phosphates (CYTH). The role of the lectin domain in the determination of the substrate specificity of these enzymes suggests that Eg-ppGalNAc-T1 would be involved in the glycosylation of a special type of substrate. Analysis of the tissue distribution by in situ hybridization and immunohistochemistry revealed that this transferase is expressed in the hydatid cyst wall and the subtegumental region of larval worms. Therefore it could participate in the biosynthesis of O-glycosylated parasite proteins exposed at the interface between E. granulosus and its hosts.

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The subcellular location, maturation and response to increased plasma glucagon of Ruthenium Red-insensitive calcium-ion transport in rat liver

Biochemical Journal ◽

10.1042/bj1741021 ◽

1978 ◽

Vol 174 (3) ◽

pp. 1021-1030 ◽

Cited By ~ 33

Author(s):

Fyfe L. Bygrave ◽

Charmaine J. Tranter

Keyword(s):

Rat Liver ◽

Microsomal Fraction ◽

Adult Life ◽

Subcellular Location ◽

Maximal Activity ◽

Mitochondrial Fraction ◽

Ruthenium Red ◽

Maximal Response ◽

Transport Activity ◽

Adult Rats

1. The subcellular distribution and maturation of Ruthenium Red-insensitive Ca2+ transport activity were determined in livers of rats ranging in age from 3 days pre-term to 10 weeks of adult life and compared with those of glucose 6-phosphatase, 5′-nucleotidase and Ruthenium Red-sensitive Ca2+ transport. Initial rates of Ruthenium Red-insensitive Ca2+ transport were highest in those fractions enriched in glucose 6-phosphatase, i.e. the microsomal fraction; this fraction was devoid of Ruthenium Red-sensitive Ca2+ transport activity. Although the heaviest fraction (nuclear) contained significant amounts of 5′-nucleotidase activity it was devoid of Ruthenium Red-insensitive Ca2+ transport activity. 2. Foetal rat liver contain minimal amounts of Ruthenium Red-insensitive Ca2+ transport activity, glucose 6-phosphatase and 5′-nucleotidase activities. These begin to be expressed concomitantly soon after birth; Ruthenium Red-insensitive Ca2+ transport is maximal by 3 to 4 days and remains so for up to at least 10 weeks of adult life. Glucose 6-phosphatase also reaches a peak at 3–4 days, but then rapidly decreases to approach adult values. Maximal activity of 5′-nucleotidase in the microsomal and nuclear fractions is seen about 4–6 days after birth; this enzyme activity remains increased for up to about 10 days and then falls, but not as rapidly as glucose 6-phosphatase. It is tentatively suggested that the bulk of the Ruthenium Red-insensitive Ca2+ transport is attributable to the system derived from the endoplasmic reticulum. 3. Administration of glucagon to adult rats enhances by 2–3-fold the initial rate of Ruthenium Red-insensitive Ca2+ transport in the intermediate but not the microsomal fraction. The hormone-induced effect is fully suppressed by co-administration of puromycin, is dose-dependent with half-maximal response at approx. 1μg of glucagon/100g body wt. and time-dependent exhibiting a half-maximal response about 1h after administration of the hormone. 4. Ruthenium Red-insensitive Ca2+ transport in the post-mitochondrial fraction of foetal liver also responds to the administration in situ of glucagon. The response, which also is prevented by co-administration of puromycin, is maximal in those foetuses nearing term. The suggestion is made that these effects of the hormone on Ruthenium Red-insensitive Ca2+ transport are an integral part of the physiological network in the liver cell.

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Functional Characterization of the Nep1-Like Protein Effectors of the Necrotrophic Pathogen – Alternaria brassicae

Frontiers in Microbiology ◽

10.3389/fmicb.2021.738617 ◽

2021 ◽

Vol 12 ◽

Author(s):

Deepak Duhan ◽

Shivani Gajbhiye ◽

Rajdeep Jaswal ◽

Ravindra Pal Singh ◽

Tilak Raj Sharma ◽

...

Keyword(s):

Cell Death ◽

Host Species ◽

Functional Characterization ◽

Subcellular Location ◽

Ethylene Biosynthesis ◽

Alternaria Brassicae ◽

Necrotrophic Pathogen ◽

Molecular Pattern ◽

Pathogen Associated Molecular Pattern

Alternaria brassicae is an important necrotrophic pathogen that infects the Brassicaceae family. A. brassicae, like other necrotrophs, also secretes various proteinaceous effectors and metabolites that cause cell death to establish itself in the host. However, there has been no systematic study of A. brassicae effectors and their roles in pathogenesis. The availability of the genome sequence of A. brassicae in public domain has enabled the search for effectors and their functional characterization. Nep1-like proteins (NLPs) are a superfamily of proteins that induce necrosis and ethylene biosynthesis. They have been reported from a variety of microbes including bacteria, fungi, and oomycetes. In this study, we identified two NLPs from A. brassicae viz. AbrNLP1 and AbrNLP2 and functionally characterized them. Although both AbrNLPs were found to be secretory in nature, they localized differentially inside the plant. AbrNLP2 was found to induce necrosis in both host and non-host species, while AbrNLP1 could not induce necrosis in both species. Additionally, AbrNLP2 was shown to induce pathogen-associated molecular pattern (PAMP)-triggered immunity in both host and non-host species. Overall, our study indicates that AbrNLPs are functionally and spatially (subcellular location) distinct and may play different but important roles during the pathogenesis of A. brassicae.

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Functional characterization of human brown adipose tissue metabolism

Biochemical Journal ◽

10.1042/bcj20190464 ◽

2020 ◽

Vol 477 (7) ◽

pp. 1261-1286 ◽

Cited By ~ 3

Author(s):

Marie Anne Richard ◽

Hannah Pallubinsky ◽

Denis P. Blondin

Keyword(s):

Adipose Tissue ◽

Brown Adipose Tissue ◽

Functional Characterization ◽

Human Infants ◽

Positron Emission ◽

Brown Adipose ◽

Treatment Of Obesity ◽

And Function

Brown adipose tissue (BAT) has long been described according to its histological features as a multilocular, lipid-containing tissue, light brown in color, that is also responsive to the cold and found especially in hibernating mammals and human infants. Its presence in both hibernators and human infants, combined with its function as a heat-generating organ, raised many questions about its role in humans. Early characterizations of the tissue in humans focused on its progressive atrophy with age and its apparent importance for cold-exposed workers. However, the use of positron emission tomography (PET) with the glucose tracer [18F]fluorodeoxyglucose ([18F]FDG) made it possible to begin characterizing the possible function of BAT in adult humans, and whether it could play a role in the prevention or treatment of obesity and type 2 diabetes (T2D). This review focuses on the in vivo functional characterization of human BAT, the methodological approaches applied to examine these features and addresses critical gaps that remain in moving the field forward. Specifically, we describe the anatomical and biomolecular features of human BAT, the modalities and applications of non-invasive tools such as PET and magnetic resonance imaging coupled with spectroscopy (MRI/MRS) to study BAT morphology and function in vivo, and finally describe the functional characteristics of human BAT that have only been possible through the development and application of such tools.

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