scholarly journals Characterization of the Proprotein Convertase-Mediated Processing of Peroxidasin and Peroxidasin-like Protein

Antioxidants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1565
Author(s):  
Hajnal A. Kovács ◽  
Enikő Lázár ◽  
György Várady ◽  
Gábor Sirokmány ◽  
Miklós Geiszt
Keyword(s):  
Peroxidase Activity ◽  
Protein Quality ◽  
Collagen Iv ◽  
Proteolytic Processing ◽  
Proprotein Convertase ◽  
Basement Membrane Synthesis ◽  
Unknown Protein ◽  
Sequence Elements

Peroxidasin (PXDN) and peroxidasin-like protein (PXDNL) are members of the peroxidase-cyclooxygenase superfamily. PXDN functions in basement membrane synthesis by forming collagen IV crosslinks, while the function of PXDNL remains practically unknown. In this work, we characterized the post-translational proteolytic processing of PXDN and PXDNL. Using a novel knock-in mouse model, we demonstrate that the proteolytic cleavage of PXDN occurs in vivo. With the help of furin-specific siRNA we also demonstrate that the proprotein-convertase, furin participates in the proteolytic processing of PXDN. Furthermore, we demonstrate that only the proteolytically processed PXDN integrates into the extracellular matrix, highlighting the importance of the proteolysis step in PXDN’s collagen IV-crosslinking activity. We also provide multiple lines of evidence for the importance of peroxidase activity in the proteolytic processing of PXDN. Finally, we show that PXDNL does not undergo proteolytic processing, despite containing sequence elements efficiently recognized by proprotein convertases. Collectively, our observations suggest a previously unknown protein quality control during PXDN synthesis and the importance of the peroxidase activity of PXDN in this process.

scholarly journals A Conserved Biogenesis Pathway for Nucleoporins: Proteolytic Processing of a 186-Kilodalton Precursor Generates Nup98 and the Novel Nucleoporin, Nup96

1999 ◽  
Vol 144 (6) ◽  
pp. 1097-1112 ◽  
Author(s):  
Beatriz M.A. Fontoura ◽  
Günter Blobel ◽  
Michael J. Matunis
Keyword(s):  
Proteolytic Cleavage ◽  
Proteolytic Processing ◽  
Precursor Protein ◽  
Nuclear Pore ◽  
Biochemical Fractionation ◽  
Alternatively Spliced ◽  
Functional Analyses ◽  
Related Proteins

The mammalian nuclear pore complex (NPC) is comprised of ∼50 unique proteins, collectively known as nucleoporins. Through fractionation of rat liver nuclei, we have isolated >30 potentially novel nucleoporins and have begun a systematic characterization of these proteins. Here, we present the characterization of Nup96, a novel nucleoporin with a predicted molecular mass of 96 kD. Nup96 is generated through an unusual biogenesis pathway that involves synthesis of a 186-kD precursor protein. Proteolytic cleavage of the precursor yields two nucleoporins: Nup98, a previously characterized GLFG-repeat containing nucleoporin, and Nup96. Mutational and functional analyses demonstrate that both the Nup98-Nup96 precursor and the previously characterized Nup98 (synthesized independently from an alternatively spliced mRNA) are proteolytically cleaved in vivo. This biogenesis pathway for Nup98 and Nup96 is evolutionarily conserved, as the putative Saccharomyces cerevisiae homologues, N-Nup145p and C-Nup145p, are also produced through proteolytic cleavage of a precursor protein. Using immunoelectron microscopy, Nup96 was localized to the nucleoplasmic side of the NPC, at or near the nucleoplasmic basket. The correct targeting of both Nup96 and Nup98 to the nucleoplasmic side of the NPC was found to be dependent on proteolytic cleavage, suggesting that the cleavage process may regulate NPC assembly. Finally, by biochemical fractionation, a complex containing Nup96, Nup107, and at least two Sec13- related proteins was identified, revealing that a major sub-complex of the NPC is conserved between yeast and mammals.

scholarly journals Characterization of the Unusual BidirectionalvesPromoters Driving VESA1 Expression and Associated with Antigenic Variation in Babesia bovis

2012 ◽  
Vol 11 (3) ◽  
pp. 260-269 ◽  
Author(s):  
Xinyi Wang ◽  
Yu-Ping Xiao ◽  
Anne Bouchut ◽  
Basima Al-Khedery ◽  
Hongbin Wang ◽  
...  
Keyword(s):  
Antigenic Variation ◽  
Initial Step ◽  
Firefly Luciferase ◽  
Bidirectional Promoter ◽  
Babesia Bovis ◽  
Regulatory Sequences ◽  
Content Type ◽  
Sequence Elements

ABSTRACTRapid clonal antigenic variation inBabesia bovisinvolves thevarianterythrocytesurfaceantigen-1 (VESA1) protein expressed on the infected-erythrocyte surface. Because of the significance of this heterodimeric protein for demonstrated mechanisms of parasite survival and virulence, there is a need to understand how expression of thevesmultigene family encoding this protein is controlled. As an initial step toward this goal, we present here initial characterization of thevespromoter driving transcription of VESA1a and -1b subunits. A series of transfection constructs containing various sequence elements from thein vivolocus of activevestranscription (LAT) were used to drive expression of the firefly luciferase gene in a dual luciferase-normalized assay. The results of this approach reveal the presence of two bidirectional promoter activities within the 434-bp intergenic region (IGr), influenced by putative regulatory sequences embedded within the flankingves1α andves1β genes. Repressor-like effects on the apposing gene were observed for intron 1 of bothves1α andves1β. This effect is apparently not dependent upon intronic promoter activity and acts only incis. The expression of genes within thevesfamily is likely modulated by local elements embedded withinvescoding sequences outside the intergenic promoter region in concert with chromatin modifications. These results provide a framework to help us begin to understand gene regulation during antigenic variation inB. bovis.

scholarly journals In Vivo Methods to Study Protein–Protein Interactions as Key Players in Mycobacterium Tuberculosis Virulence

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 173 ◽  
Author(s):  
Veyron-Churlet ◽  
Locht
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Interactions ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Unknown Protein ◽  
Protein Functions ◽  
Transient Interactions ◽  
Protein Complementation

Studies on protein–protein interactions (PPI) can be helpful for the annotation of unknown protein functions and for the understanding of cellular processes, such as specific virulence mechanisms developed by bacterial pathogens. In that context, several methods have been extensively used in recent years for the characterization of Mycobacterium tuberculosis PPI to further decipher tuberculosis (TB) pathogenesis. This review aims at compiling the most striking results based on in vivo methods (yeast and bacterial two-hybrid systems, protein complementation assays) for the specific study of PPI in mycobacteria. Moreover, newly developed methods, such as in-cell native mass resonance and proximity-dependent biotinylation identification, will have a deep impact on future mycobacterial research, as they are able to perform dynamic (transient interactions) and integrative (multiprotein complexes) analyses.

scholarly journals In Vivo Methods to Study Protein-Protein Interactions as Key Players in Mycobacterium tuberculosis Virulence

2019 ◽  
Author(s):  
Romain Veyron-Churlet ◽  
Camille Locht
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Interactions ◽  
Protein Function ◽  
Protein Protein Interactions ◽  
Cellular Processes ◽  
Unknown Protein ◽  
Transient Interactions ◽  
Protein Complementation

Studies on Protein-Protein interactions (PPI) can be helpful for the annotation of unknown protein function and for the understanding of cellular processes, such as specific virulence mechanisms developed by bacterial pathogens. In that context, several methods have been extensively used in recent years for the characterization of Mycobacterium tuberculosis PPI to further decipher TB pathogenesis. This review aims at compiling the most striking results based on in vivo methods (yeast and bacterial two-hybrid systems, protein complementation assays) for the specific study of PPI in mycobacteria. Moreover, newly developed methods, such as in-cell native mass resonance and proximity-dependent biotinylation identification, will have a deep impact on future mycobacterial research, as they are able to perform dynamic (transient interactions) and integrative (multiprotein complexes) analyses.

scholarly journals Detoxification of DNA hydroperoxide by glutathione transferases and the purification and characterization of glutathione transferases of the rat liver nucleus

1988 ◽  
Vol 254 (3) ◽  
pp. 841-845 ◽  
Author(s):  
K H Tan ◽  
D J Meyer ◽  
N Gillies ◽  
B Ketterer
Keyword(s):  
Rat Liver ◽  
Peroxidase Activity ◽  
Glutathione Transferases ◽  
Supernatant Fraction ◽  
Liver Nucleus ◽  
Purification And Characterization ◽  
Soluble Supernatant

DNA peroxidized by exposure to ionizing radiation in the presence of oxygen is a substrate for the Se-independent GSH peroxidase activity of several GSH transferases, GSH transferases 5-5, 3-3 and 4-4 being the most active in the rat liver soluble supernatant fraction (500, 35 and 20 nmol/min per mg of protein respectively) and GSH transferases mu and pi the most active, so far found, in the human liver soluble supernatant fraction (80 and 10 nmol/min per mg respectively). Although the GSH transferase content of the rat nucleus was found to be much lower than that of the soluble supernatant, nuclear GSH transferases are likely to be more important in the detoxification of DNA hydroperoxide produced in vivo. Two nuclear fractions were studied, one extracted with 0.075 M-saline/0.025 M-EDTA, pH 8.0, and the other extracted from the residue with 8.5 M-urea. The saline/EDTA fraction contained subunits 1, 2, 3, 4 and a novel subunit, similar but not identical to 5, provisionally referred to as 5*, in the proportions 40:25:5:5:25 respectively. The 8.5 M-urea-extracted fraction contained principally subunit 5* together with a small amount of subunit 6 in the proportion 95:5 respectively. GSH transferase 5*-5* purified from the 8.5 M-urea extract has the highest activity towards DNA hydroperoxide of any GSH transferase so far studied (1.5 mumol/min per mg). A Se-dependent GSH peroxidase fraction from rat liver was also active towards DNA hydroperoxide; however, since this enzyme accounts for only 14% of the GSH peroxidase activity detectable in the nucleus, GSH transferases may be the more important source of this activity. The possible role of GSH transferases, in particular GSH transferase 5*-5*, in DNA repair is discussed.

scholarly journals Expression of Murine Coronavirus Recombinant Papain-Like Proteinase: Efficient Cleavage Is Dependent on the Lengths of both the Substrate and the Proteinase Polypeptides

1999 ◽  
Vol 73 (4) ◽  
pp. 2658-2666 ◽  
Author(s):  
Henry Teng ◽  
Josefina D. Piñón ◽  
Susan R. Weiss
Keyword(s):  
Hepatitis Virus ◽  
In Vitro Transcription ◽  
Proteolytic Processing ◽  
Replicase Gene ◽  
Cleavage Activity ◽  
Viral Sequences ◽  
Rna Polymerase Promoter

ABSTRACT Proteolytic processing of the replicase gene product of mouse hepatitis virus (MHV) is essential for viral replication. In MHV strain A59 (MHV-A59), the replicase gene encodes two predicted papain-like proteinase (PLP) domains, PLP-1 and PLP-2. Previous work using viral polypeptide substrates synthesized by in vitro transcription and translation from the replicase gene demonstrated both cisand trans cleavage activities for PLP-1. We have cloned and overexpressed the PLP-1 domain in Escherichia coli by using a T7 RNA polymerase promoter system or as a maltose-binding protein (MBP) fusion protein. With both overexpression systems, the recombinant PLP-1 exhibited trans cleavage activity when incubated with in vitro-synthesized viral polypeptide substrates. Subsequent characterization of the recombinant PLP-1 revealed that in vitrotrans cleavage is more efficient at 22°C than at higher temperatures. Using substrates of increasing lengths, we observed efficient cleavage by PLP-1 requires a substrate greater than 69 kDa. In addition, when PLP-1 was expressed as a polypeptide that included additional viral sequences at the carboxyl terminus of the predicted PLP-1 domain, a fivefold increase in proteolytic activity was observed. The data presented here support previous data suggesting that in vitro and in vivo cleavage of the ORF 1a polyprotein by PLP-1 can occur in both in cis and in trans. In contrast to the cleavage activity demonstrated for PLP-1, no in vitro cleavage incis or in trans could be detected with PLP-2 expressed either as a polypeptide, including flanking viral sequences, or as an MBP fusion enzyme.

Functional characterization of human brown adipose tissue metabolism

2020 ◽  
Vol 477 (7) ◽  
pp. 1261-1286 ◽  
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.

Comparison of Immunological and Functional Assays for Measurement of Soluble Fibrin

1995 ◽  
Vol 74 (02) ◽  
pp. 673-679 ◽  
Author(s):  
C E Dempfle ◽  
S A Pfitzner ◽  
M Dollman ◽  
K Huck ◽  
G Stehle ◽  
...  
Keyword(s):  
Venous Thrombosis ◽  
Low Grade ◽  
Plasma Samples ◽  
Normal Range ◽  
Soluble Fibrin ◽  
Discriminating Power ◽  
Fibrin Monomer ◽  
Cerebral Ischemic

SummaryVarious assays have been developed for quantitation of soluble fibrin or fibrin monomer in clinical plasma samples, since this parameter directly reflects in vivo thrombin action on fibrinogen. Using plasma samples from healthy blood donors, patients with cerebral ischemic insult, patients with septicemia, and patients with venous thrombosis, we compared two immunologic tests using monoclonal antibodies against fibrin-specific neo-epitopes, and two functional tests based on the cofactor activity of soluble fibrin complexes in tPA-induced plasminogen activation. Test A (Enzymun®-Test FM) showed the best discriminating power among normal range and pathological samples. Test B (Fibrinostika® soluble fibrin) clearly separated normal range from pathological samples, but failed to discriminate among samples from patients with low grade coagulation activation in septicemia, and massive activation in venous thrombosis. Functional test C (Fibrin monomer test Behring) displayed good discriminating power between normal and pathological range samples, and correlated with test A (r = 0.61), whereas assay D (Coa-Set® Fibrin monomer) showed little discriminating power at values below 10 μg/ml and little correlation with other assays. Standardization of assays will require further characterization of analytes detected.

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