scholarly journals Mammalian NADH diphosphatases of the Nudix family: cloning and characterization of the human peroxisomal NUDT12 protein

2003 ◽  
Vol 374 (2) ◽  
pp. 329-335 ◽  
Author(s):  
Salama R. ABDELRAHEIM ◽  
David G. SPILLER ◽  
Alexander G. McLENNAN
Keyword(s):  
Fluorescent Protein ◽  
Nudix Hydrolase ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Diadenosine Triphosphate ◽  
Green Fluorescent ◽  
Cytoplasmic Structures

The human NUDT12 Nudix hydrolase has been expressed in insect cells from a baculovirus vector as a His-tagged recombinant protein. In vitro, it efficiently hydrolyses NAD(P)H to NMNH and AMP (2′,5′-ADP), and diadenosine diphosphate to AMP. It also has activity towards NAD(P)+, ADP-ribose and diadenosine triphosphate. Km values for NADH, NADPH and NAD+ are 11, 16 and 190 μM and kcat values are 11, 16 and 10.5 s−1 respectively. Thus, like other NADH diphosphatases of the Nudix family, NUDT12 has a marked substrate preference for the reduced nicotinamide nucleotides. Optimal activity was supported by 50 μM Mn2+ ions in vitro, with 3-fold lower activity at 0.4 mM Mg2+. Expression of NUDT12 as a C-terminal fusion to green fluorescent protein revealed that it was targeted to peroxisomes by the C-terminal tripeptide PNL acting as a novel type 1 peroxisomal targeting signal. Deletion of PNL resulted in diffuse cellular fluorescence. In addition, C-terminal, but not N-terminal, fusions with or without the PNL signal accumulated in large, unidentified cytoplasmic structures. NUDT12 may act to regulate the concentration of peroxisomal nicotinamide nucleotide cofactors required for oxidative metabolism in this organelle.

scholarly journals Functional Analyses of a Putative, Membrane-Bound, Peroxisomal Protein Import Mechanism from the Apicomplexan Protozoan Toxoplasma gondii

Genes ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 434
Author(s):  
Alison Mbekeani ◽  
Will Stanley ◽  
Vishal Kalel ◽  
Noa Dahan ◽  
Einat Zalckvar ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Protein Import ◽  
Fluorescent Protein ◽  
Metabolic Energy ◽  
Peroxisomal Protein ◽  
Genes Encoding ◽  
Peroxisomal Targeting ◽  
Membrane Bound ◽  
Green Fluorescent

Peroxisomes are central to eukaryotic metabolism, including the oxidation of fatty acids—which subsequently provide an important source of metabolic energy—and in the biosynthesis of cholesterol and plasmalogens. However, the presence and nature of peroxisomes in the parasitic apicomplexan protozoa remains controversial. A survey of the available genomes revealed that genes encoding peroxisome biogenesis factors, so-called peroxins (Pex), are only present in a subset of these parasites, the coccidia. The basic principle of peroxisomal protein import is evolutionarily conserved, proteins harbouring a peroxisomal-targeting signal 1 (PTS1) interact in the cytosol with the shuttling receptor Pex5 and are then imported into the peroxisome via the membrane-bound protein complex formed by Pex13 and Pex14. Surprisingly, whilst Pex5 is clearly identifiable, Pex13 and, perhaps, Pex14 are apparently absent from the coccidian genomes. To investigate the functionality of the PTS1 import mechanism in these parasites, expression of Pex5 from the model coccidian Toxoplasma gondii was shown to rescue the import defect of Pex5-deleted Saccharomyces cerevisiae. In support of these data, green fluorescent protein (GFP) bearing the enhanced (e)PTS1 known to efficiently localise to peroxisomes in yeast, localised to peroxisome-like bodies when expressed in Toxoplasma. Furthermore, the PTS1-binding domain of Pex5 and a PTS1 ligand from the putatively peroxisome-localised Toxoplasma sterol carrier protein (SCP2) were shown to interact in vitro. Taken together, these data demonstrate that the Pex5–PTS1 interaction is functional in the coccidia and indicate that a nonconventional peroxisomal import mechanism may operate in the absence of Pex13 and Pex14.

In Vitro and In Vivo Characterization of Recombinant Ebola Viruses Expressing Enhanced Green Fluorescent Protein

2007 ◽  
Vol 196 (s2) ◽  
pp. S313-S322 ◽  
Author(s):  
Hideki Ebihara ◽  
Steven Theriault ◽  
Gabriele Neumann ◽  
Judie B. Alimonti ◽  
Joan B. Geisbert ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Green Fluorescent ◽  
In Vivo Characterization

scholarly journals C-terminal tripeptide Ser-Asn-Leu (SNL) of human D-aspartate oxidase is a functional peroxisome-targeting signal

1998 ◽  
Vol 336 (2) ◽  
pp. 367-371 ◽  
Author(s):  
Leen AMERY ◽  
Chantal BREES ◽  
Myriam BAES ◽  
Chiaki SETOYAMA ◽  
Retsu MIURA ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Amino Acid ◽  
Fluorescent Protein ◽  
Consensus Sequence ◽  
C Terminus ◽  
Targeting Signal ◽  
Fluorescent Pattern ◽  
Green Fluorescent ◽  
Positively Charged

The functionality of the C-terminus (Ser-Asn-Leu; SNL) of human d-aspartate oxidase, an enzyme proposed to have a role in the inactivation of synaptically released d-aspartate, as a peroxisome-targeting signal (PTS1) was investigated in vivoand in vitro. Bacterially expressed human d-aspartate oxidase was shown to interact with the human PTS1-binding protein, peroxin protein 5 (PEX5p). Binding was gradually abolished by carboxypeptidase treatment of the oxidase and competitively inhibited by a Ser-Lys-Leu (SKL)-containing peptide. After transfection of mouse fibroblasts with a plasmid encoding green fluorescent protein (GFP) extended by PKSNL (the C-terminal pentapeptide of the oxidase), a punctate fluorescent pattern was evident. The modified GFP co-localized with peroxisomal thiolase as shown by indirect immunofluorescence. On transfection in fibroblasts lacking PEX5p receptor, GFP–PKSNL staining was cytosolic. Peroxisomal import of GFP extended by PGSNL (replacement of the positively charged fourth-last amino acid by glycine) seemed to be slower than that of GFP–PKSNL, whereas extension by PKSNG abolished the import of the modified GFP. Taken together, these results indicate that SNL, a tripeptide not fitting the PTS1 consensus currently defined in mammalian systems, acts as a functional PTS1 in mammalian systems, and that the consensus sequence, based on this work and that of other groups, has to be broadened to (S/A/C/K/N)-(K/R/H/Q/N/S)-L.

scholarly journals Preparation and Characterization of Gelatin-Based Mucoadhesive Nanocomposites as Intravesical Gene Delivery Scaffolds

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Ching-Wen Liu ◽  
Li-Ching Chang ◽  
Kai-Jen Lin ◽  
Tsan-Jung Yu ◽  
Ching-Chung Tsai ◽  
...  
Keyword(s):  
Gene Delivery ◽  
Fluorescent Protein ◽  
Crosslinking Density ◽  
Intravesical Instillation ◽  
Bladder Compliance ◽  
Release Studies ◽  
Green Fluorescent

This study aimed to develop optimal gelatin-based mucoadhesive nanocomposites as scaffolds for intravesical gene delivery to the urothelium. Hydrogels were prepared by chemically crosslinking gelatin A or B with glutaraldehyde. Physicochemical and delivery properties including hydration ratio, viscosity, size, yield, thermosensitivity, and enzymatic degradation were studied, and scanning electron microscopy (SEM) was carried out. The optimal hydrogels (H), composed of 15% gelatin A175, displayed an 81.5% yield rate, 87.1% hydration ratio, 42.9 Pa·s viscosity, and 125.8 nm particle size. The crosslinking density of the hydrogels was determined by performing pronase degradation and ninhydrin assays.In vitrolentivirus (LV) release studies involving p24 capsid protein analysis in 293T cells revealed that hydrogels containing lentivirus (H-LV) had a higher cumulative release than that observed for LV alone (3.7-, 2.3-, and 2.3-fold at days 1, 3, and 5, resp.). Lentivirus from lentivector constructed green fluorescent protein (GFP) was then entrapped in hydrogels (H-LV-GFP). H-LV-GFP showed enhanced gene delivery in AY-27 cellsin vitroand to rat urothelium by intravesical instillationin vivo. Cystometrogram showed mucoadhesive H-LV reduced peak micturition and threshold pressure and increased bladder compliance. In this study, we successfully developed first optimal gelatin-based mucoadhesive nanocomposites as intravesical gene delivery scaffolds.

scholarly journals The sorting sequence of the peroxisomal integral membrane protein PMP47 is contained within a short hydrophilic loop.

1996 ◽  
Vol 133 (2) ◽  
pp. 269-280 ◽  
Author(s):  
J M Dyer ◽  
J A McNew ◽  
J M Goodman
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
The Matrix ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Green Fluorescent ◽  
Necessary And Sufficient ◽  
Hydrophilic Loop

No targeting sequence for peroxisomal integral membrane proteins has yet been identified. We have previously shown that a region of 67 amino acids is necessary to target Pmp47, a protein that spans the membrane six times, to peroxisomes. This region comprises two membrane spans and the intervening loop. We now demonstrate that the 20 amino acid loop, which is predicted to face the matrix, is both necessary and sufficient for peroxisomal targeting. Sufficiency was demonstrated with both chloramphenicol acetyltransferase and green fluorescent protein as carriers. There is a cluster of basic amino acids in the middle of the loop that we predict protrudes from the membrane surface into the matrix by a flanking stem structure. We show that the targeting signal is composed of this basic cluster and a block of amino acids immediately down-stream from it.

scholarly journals Alf1p, a CLIP-170 Domain-containing Protein, Is Functionally and Physically Associated with α-Tubulin

1999 ◽  
Vol 144 (1) ◽  
pp. 113-124 ◽  
Author(s):  
Becket Feierbach ◽  
Eva Nogales ◽  
Kenneth H. Downing ◽  
Tim Stearns
Keyword(s):  
Fluorescent Protein ◽  
Null Alleles ◽  
Microtubule Associated Proteins ◽  
Cytoplasmic Face ◽  
Associated Proteins ◽  
Green Fluorescent ◽  
Β Tubulin

Tubulin is a heterodimer of α- and β-tubulin polypeptides. Assembly of the tubulin heterodimer in vitro requires the CCT chaperonin complex, and a set of five proteins referred to as the tubulin cofactors (Tian, F., Y. Huang, H. Rommelaere, J. Vandekerckhove, C. Ampe, and N.J. Cowan. 1996. Cell. 86:287–296; Tian, G., S.A. Lewis, B. Feierbach, T. Stearns, H. Rommelaere, C. Ampe, and N.J. Cowan. 1997. J. Cell Biol. 138:821–832). We report the characterization of Alf1p, the yeast ortholog of mammalian cofactor B. Alf1p interacts with α-tubulin in both two-hybrid and immunoprecipitation assays. Alf1p and cofactor B contain a single CLIP-170 domain, which is found in several microtubule-associated proteins. Mutation of the CLIP-170 domain in Alf1p disrupts the interaction with α-tubulin. Mutations in α-tubulin that disrupt the interaction with Alf1p map to a domain on the cytoplasmic face of α-tubulin; this domain is distinct from the region of interaction between α-tubulin and β-tubulin. Alf1p-green fluorescent protein (GFP) is able to associate with microtubules in vivo, and this localization is abolished either by mutation of the CLIP-170 domain in Alf1p, or by mutation of the Alf1p-binding domain in α-tubulin. Analysis of double mutants constructed between null alleles of ALF1 and PAC2, which encodes the other yeast α-tubulin cofactor, suggests that Alf1p and Pac2p act in the same pathway leading to functional α-tubulin. The phenotype of overexpression of ALF1 suggests that Alf1p can act to sequester α-tubulin from interaction with β-tubulin, raising the possibility that it plays a regulatory role in the formation of the tubulin heterodimer.

scholarly journals Peroxisome Degradation by Microautophagy in Pichia pastoris: Identification of Specific Steps and Morphological Intermediates

1998 ◽  
Vol 141 (3) ◽  
pp. 625-636 ◽  
Author(s):  
Yasuyoshi Sakai ◽  
Antonius Koller ◽  
Linda K. Rangell ◽  
Gilbert A. Keller ◽  
Suresh Subramani
Keyword(s):  
Green Fluorescent Protein ◽  
Pichia Pastoris ◽  
Fluorescent Protein ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Complementation Groups ◽  
Green Fluorescent ◽  
Kinetics Of ◽  
Pyridinium Dibromide ◽  
Shed Light

We used the dye N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenylhexatrienyl) pyridinium dibromide (FM4-64) and a fusion protein, consisting of the green fluorescent protein appended to the peroxisomal targeting signal, Ser-Lys-Leu (SKL), to label the vacuolar membrane and the peroxisomal matrix, respectively, in living Pichia pastoris cells and followed by fluorescence microscopy the morphological and kinetic intermediates in the vacuolar degradation of peroxisomes by microautophagy and macroautophagy. Structures corresponding to the intermediates were also identified by electron microscopy. The kinetics of appearance and disappearance of these intermediates is consistent with a precursor–product relationship between intermediates, which form the basis of a model for microautophagy. Inhibitors affecting different steps of microautophagy did not impair peroxisome delivery to the vacuole via macroautophagy, although inhibition of vacuolar proteases affected the final vacuolar degradation of green fluorescent protein (S65T mutant version [GFP])-SKL via both autophagic pathways. P. pastoris mutants defective in peroxisome microautophagy (pag mutants) were isolated and characterized for the presence or absence of the intermediates. These mutants, comprising 6 complementation groups, support the model for microautophagy. Our studies indicate that the microautophagic degradation of peroxisomes proceeds via specific intermediates, whose generation and/or processing is controlled by PAG gene products, and shed light on the poorly understood phenomenon of peroxisome homeostasis.

scholarly journals Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p

2000 ◽  
Vol 346 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Rachel K. SZILARD ◽  
Richard A. RACHUBINSKI
Keyword(s):  
Yarrowia Lipolytica ◽  
Tetratricopeptide Repeat ◽  
Binding Assays ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Tetratricopeptide Repeat Domain ◽  
Tpr Domain ◽  
Peroxisomal Targeting Signal

Peroxins are proteins required for peroxisome assembly and are encoded by the PEX genes. The Yarrowia lipolytica pex5-1 mutant fails to import a subset of peroxisomal matrix proteins, including those with a type 1 peroxisomal targeting signal (PTS1). Pex5p family members interact with a PTS1 through their characteristic tetratricopeptide repeat (TPR) domain. We used binding assays in vitro to investigate the nature of the association of Y. lipolytica Pex5p (YlPex5p) with the PTS1 signal. A purified recombinant YlPex5p fusion protein interacted specifically, directly and autonomously with a protein terminating in a PTS1. Wild-type YlPex5p translated in vitro recognized functional PTS1s specifically. This activity is abrogated by the substitution of an aspartic residue for a conserved glycine residue in the TPR domain (G455D) of YlPex5p encoded by the pex5-1 allele. Deletion analysis demonstrated that an intact TPR domain of YlPex5p is necessary but not sufficient for both interaction with a PTS1 and functional complementation of a strain lacking YlPex5p.

scholarly journals Identification of a Type 1 Peroxisomal Targeting Signal in a Viral Protein and Demonstration of Its Targeting to the Organelle

2002 ◽  
Vol 76 (5) ◽  
pp. 2543-2547 ◽  
Author(s):  
K. V. K. Mohan ◽  
I. Som ◽  
C. D. Atreya
Keyword(s):  
Fluorescent Protein ◽  
Morphological Changes ◽  
Consensus Sequence ◽  
Amino Acid Sequences ◽  
Cytoplasmic Staining ◽  
Cellular Expression ◽  
Peroxisomal Targeting ◽  
Targeting Signal ◽  
Peroxisomal Targeting Signal

ABSTRACT Peroxisomes are unimembrane, respiratory organelles of the cell. Transport of cellular proteins to the peroxisomal matrix requires a type 1 peroxisomal targeting signal (PTS1) which essentially constitutes a tripeptide from the consensus sequence S/T/A/G/C/N-K/R/H-L/I/V/M/A/F/Y. Although PTS-containing proteins have been identified in eukaryotes, prokaryotes, and parasites, viral proteins with such signals have not been identified so far. We report here the first instance of a virus, the rotavirus, which causes infantile diarrhea worldwide, containing a functional C-terminal PTS1 in one of its proteins (VP4). Analysis of 153 rotavirus VP4-deduced amino acid sequences identified five groups of conserved C-terminal PTS1 tripeptide sequences (SKL, CKL, GKL, CRL, and CRI), of which CRL is represented in approximately 62% of the sequences. Infection of cells by a CRL-containing representative rotavirus (SA11 strain) and confocal immunofluorescence analysis revealed colocalization of VP4 with peroxisomal markers and morphological changes of peroxisomes. Further, transient cellular expression of green fluorescent protein (GFP)-fused VP4CRL resulted in transport of VP4 to peroxisomes, whereas the chimera lacking the PTS1 signal, GFP-VP4ΔCRL, resulted in diffuse cytoplasmic staining, suggesting a CRL-dependent targeting of the protein. The present study therefore demonstrates hitherto unreported organelle involvement, specifically of the peroxisomes, in rotaviral infections as demonstrated by using the SA11 strain of rotavirus and opens a new line of investigation toward understanding viral pathogenesis and disease mechanisms.

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