scholarly journals Association and release of the major intrinsic membrane glycoprotein from peripheral nerve myelin

1985 ◽  
Vol 228 (1) ◽  
pp. 43-54 ◽  
Author(s):  
J F Poduslo ◽  
J K Yao
Keyword(s):  
Membrane Protein ◽  
Peripheral Nerve ◽  
Gamma Globulin ◽  
Protein A ◽  
Supernatant Fraction ◽  
Radioactive Label ◽  
Lower Phase ◽  
Hydrophobic Domain ◽  
Successive Extraction

Hypo-osmotic homogenization of the endoneurium from the adult-rat sciatic nerve and subsequent evaluation of the 197 000 g aqueous supernatant by sodium dodecyl sulphate pore-gradient electrophoresis (SDS-p.g.e.) revealed a release of the major glycoprotein (P0) (29 000 Mr) from peripheral nerve myelin. Immunological verification of the presence of this asparagine-linked glycoprotein in the aqueous supernatant was obtained by immune overlay after SDS-p.g.e. and electrophoretic transfer to nitrocellulose using anti-P0 gamma-globulin followed by autoradiographic detection with 125I-protein A. A comparison of successive hypo- and iso-osmotic extractions of the endoneurium revealed that the hypo-osmotic extraction released increasing amounts of P0 into the supernatant fraction, whereas the iso-osmotic treatment revealed lower levels of P0 extracted from the myelin and lesser amounts with each successive extraction. Three successive hypo-osmotic extractions resulted in a 2.0-, 2.9-, and 9.5-fold increase in the amount of P0 released compared with the successive iso-osmotic extractions. Although these results suggest that this major myelin glycoprotein has properties similar to those of extrinsic membrane proteins, temperature-dependent phase-partitioning experiments with Triton X-114 revealed that this glycoprotein is recovered in the detergent-enriched lower phase. These results indicate that this major myelin glycoprotein is an amphipathic integral membrane protein with a distinct hydrophobic domain and yet has solubility characteristics typical of an extrinsic membrane protein. P0 labelled in vitro with [3H]mannose could be immunoprecipitated from the aqueous supernatant with anti-P0 gamma-globulin by centrifugation at 197000g without the addition of second antibody or protein A. Analysis of such an immune precipitate after incorporation in vitro with [14C]acetate to label endoneurial lipids revealed that all major endoneurial lipid classes contained radioactive label, as determined by fluorography after high-performance t.l.c. The mechanism of release of this intrinsic glycoprotein from the myelin membrane, therefore, involves the osmotic-dependent formation of mixed micelles or membrane vesicles with endogenous membrane lipids.

scholarly journals Structure, biosynthesis, and localization of dipeptidyl aminopeptidase B, an integral membrane glycoprotein of the yeast vacuole.

1989 ◽  
Vol 108 (4) ◽  
pp. 1363-1373 ◽  
Author(s):  
C J Roberts ◽  
G Pohlig ◽  
J H Rothman ◽  
T H Stevens
Keyword(s):  
Membrane Protein ◽  
Secretory Pathway ◽  
Apparent Molecular Weight ◽  
Integral Membrane Protein ◽  
Restrictive Temperature ◽  
Proteolytic Activation ◽  
Hydrophobic Domain ◽  
Yeast Vacuole ◽  
Dipeptidyl Aminopeptidase

We have characterized the structure, biogenesis, and localization of dipeptidyl aminopeptidase B (DPAP B), a membrane protein of the yeast vacuole. An antibody specific for DPAP B recognizes a 120-kD glycoprotein in yeast that behaves like an integral membrane protein in that it is not removed from membranes by high pH Na2CO3 treatment. Inspection of the deduced amino acid sequence of DPAP B reveals a hydrophobic domain near the NH2 terminus that could potentially span a lipid bilayer. The in vitro enzymatic activity and apparent molecular weight of DPAP B are unaffected by the allelic state of PEP4, a gene essential for the proteolytic activation of a number of soluble vacuolar hydrolases. DPAP B is synthesized as a glycosylated precursor that is converted to the mature 120-kD species by carbohydrate addition. The precursor form of DPAP B accumulates in sec mutants (Novick, P., C. Field, and R. Schekman. 1980. Cell. 21:205-215) that are blocked at the ER (sec18) or Golgi apparatus (sec7), but not at secretory vesicles (sec1). Immunolocalization of DPAP B in wild-type or sec1 mutant cells shows that the protein resides in the vacuolar membrane. However, it is present in non-vacuolar compartments in sec18 and sec7 cells, confirming that the delivery of DPAP B is blocked in these mutants. Interestingly, DPAP B appears to stain the nuclear envelope in a sec18 mutant, which is consistent with the accumulation of DPAP B in the ER membrane at the restrictive temperature. These results suggest that soluble and membrane-bound vacuolar proteins use the same stages of the secretory pathway for their transport.

scholarly journals Virulence of the Melioidosis Pathogen Burkholderia pseudomallei Requires the Oxidoreductase Membrane Protein DsbB

2018 ◽  
Vol 86 (5) ◽  
Author(s):  
Róisín M. McMahon ◽  
Philip M. Ireland ◽  
Derek S. Sarovich ◽  
Guillaume Petit ◽  
Christopher H. Jenkins ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Burkholderia Pseudomallei ◽  
Protein A ◽  
Genomic Analysis ◽  
Core Gene ◽  
Content Type ◽  
Redox Biology

ABSTRACT The naturally antibiotic-resistant bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a disease with stubbornly high mortality and a complex, protracted treatment regimen. The worldwide incidence of melioidosis is likely grossly underreported, though it is known to be highly endemic in northern Australia and Southeast Asia. Bacterial disulfide bond (DSB) proteins catalyze the oxidative folding and isomerization of disulfide bonds in substrate proteins. In the present study, we demonstrate that B. pseudomallei membrane protein disulfide bond protein B (BpsDsbB) forms a functional redox relay with the previously characterized virulence mediator B. pseudomallei disulfide bond protein A (BpsDsbA). Genomic analysis of diverse B. pseudomallei clinical isolates demonstrated that dsbB is a highly conserved core gene. Critically, we show that DsbB is required for virulence in B. pseudomallei . A panel of B. pseudomallei dsbB deletion strains (K96243, 576, MSHR2511, MSHR0305b, and MSHR5858) were phenotypically diverse according to the results of in vitro assays that assess hallmarks of virulence. Irrespective of their in vitro virulence phenotypes, two deletion strains were attenuated in a BALB/c mouse model of infection. A crystal structure of a DsbB-derived peptide complexed with BpsDsbA provides the first molecular characterization of their interaction. This work contributes to our broader understanding of DSB redox biology and will support the design of antimicrobial drugs active against this important family of bacterial virulence targets.

scholarly journals Screening of anti-Acinetobacter baumannii phytochemicals, based on the potential inhibitory effect on OmpA and OmpW functions

2021 ◽  
Author(s):  
Shahab Shahryari ◽  
Parvin Mohammadnejad ◽  
Kambiz Akbari Noghabi
Keyword(s):  
Membrane Protein ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Simulation Analysis ◽  
Protein A ◽  
Binding Pocket ◽  
Dimensional Structure

Therapeutic options, including last-line or combined antibiotic therapies for multi-drug resistant (MDR) strains of Acinetobacter baumannii are ineffective. The outer membrane protein A (OmpA) and outer membrane protein W (OmpW) are two porins known for their different cellular functions. Identification of natural compounds with the potentials to block these putative porins can attenuate the growth of the bacteria and control the relating diseases. The current work aimed to screen a library of 384 phytochemicals according to their potentials to be used as a drug, and potentials to inhibit the function of OmpA and OmpW in A. baumannii. The phytocompounds were initially screened based on their physicochemical, absorption, distribution, metabolism, excretion, and toxicity (ADMET) drug-like properties. Afterward, the selected ligands were subjected to standard docking calculations against the predicted three-dimensional structure of OmpA and OmpW in A. baumannii. We identified three phytochemicals (isosakuranetin, aloe-emodin and pinocembrin) possessing appreciable binding affinity towards the selected binding pocket of OmpA and OmpW. Molecular dynamics (MD) simulation analysis confirmed the stability of the complexes. Amongst them, isosakuranetin was suggested as the best phytocompound for further in vitro and in vivo study.

scholarly journals Screening of anti- Acinetobacter baumannii phytochemicals, based on the potential inhibitory effect on OmpA and OmpW functions

2021 ◽  
Vol 8 (8) ◽  
pp. 201652
Author(s):  
Shahab Shahryari ◽  
Parvin Mohammadnejad ◽  
Kambiz Akbari Noghabi
Keyword(s):  
Membrane Protein ◽  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Simulation Analysis ◽  
Protein A ◽  
Dynamics Simulation ◽  
Dimensional Structure

Therapeutic options including last-line or combined antibiotic therapies for multi-drug-resistant strains of Acinetobacter baumannii are ineffective. The outer membrane protein A (OmpA) and outer membrane protein W (OmpW) are two porins known for their different cellular functions. Identification of natural compounds with the potentials to block these putative porins can attenuate the growth of the bacteria and control the relating diseases. The current work aimed to screen a library of 384 phytochemicals according to their potentials to be used as a drug, and potentials to inhibit the function of OmpA and OmpW in A. baumannii . The phytocompounds were initially screened based on their physico-chemical, absorption, distribution, metabolism, excretion and toxicity (ADMET) drug-like properties. Afterwards, the selected ligands were subjected to standard docking calculations against the predicted three-dimensional structure of OmpA and OmpW in A. baumannii . We identified three phytochemicals (isosakuranetin, aloe-emodin and pinocembrin) possessing appreciable binding affinity towards the selected binding pocket of OmpA and OmpW. Molecular dynamics simulation analysis confirmed the stability of the complexes. Among them, isosakuranetin was suggested as the best phytocompound for further in vitro and in vivo study.

scholarly journals VIP21-caveolin, a membrane protein constituent of the caveolar coat, oligomerizes in vivo and in vitro.

1995 ◽  
Vol 6 (7) ◽  
pp. 911-927 ◽  
Author(s):  
S Monier ◽  
R G Parton ◽  
F Vogel ◽  
J Behlke ◽  
A Henske ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Molecular Mass ◽  
Sodium Dodecyl ◽  
High Molecular Mass ◽  
Hydrophobic Domain ◽  
Cytoplasmic Surface ◽  
Triton X ◽  
Flanking Regions

VIP21-caveolin is a membrane protein, proposed to be a component of the striated coat covering the cytoplasmic surface of caveolae. To investigate the biochemical composition of the caveolar coat, we used our previous observation that VIP21-caveolin is present in large complexes and insoluble in the detergents CHAPS or Triton X-114. The mild treatment of these insoluble structures with sodium dodecyl sulfate leads to the detection of high molecular mass complexes of approximately 200, 400, and 600 kDa. The 400-kDa complex purified to homogeneity from dog lung is shown to consist exclusive of the two isoforms of VIP21-caveolin. Pulse-chase experiments indicate that the oligomers form early after the protein is synthesized in the endoplasmic reticulum (ER). VIP21-caveolin does indeed insert into the ER membrane through the classical translocation machinery. Its hydrophobic domain adopts an unusual loop configuration exposing the N- and C-flanking regions to the cytoplasm. Similar high molecular mass complexes can be produced from the in vitro-synthesized VIP21-caveolin. The complex formation occurs only if VIP21-caveolin isoforms are properly inserted into the membrane; formation is cytosol-dependent and does not involve a vesicle fusion step. We propose that high molecular mass oligomers of VIP21-caveolin represent the basic units forming the caveolar coat. They are formed in the ER and later, between the ER and the plasma membrane, these oligomers could associate into larger detergent-insoluble structures.

Overexpression of Outer Membrane Protein A Is a Risk Factor for Mortality in Escherichia coli Bloodstream Infections

2019 ◽  
Author(s):  
Ángel Rodríguez-Villodres ◽  
Rocío Álvarez-Marín ◽  
María Antonia Pérez-Moreno ◽  
Andrea Miró-Canturri ◽  
Marco Durán Lobato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Risk Factor ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Protein A ◽  
Bloodstream Infections ◽  
Outer Membrane Protein A

scholarly journals APOBEC1 cytosine deaminase activity on single-stranded DNA is suppressed by replication protein A

2020 ◽  
Author(s):  
Lai Wong ◽  
Frederick S Vizeacoumar ◽  
Franco J Vizeacoumar ◽  
Linda Chelico
Keyword(s):  
Genomic Dna ◽  
Cytidine Deaminase ◽  
Protein A ◽  
Cytosine Deaminase ◽  
Cancer Cell Line ◽  
Replication Protein A ◽  
Lung Cancer Cell Line ◽  
Replication Protein ◽  
Cancer Evolution

Abstract Many APOBEC cytidine deaminase members are known to induce ‘off-target’ cytidine deaminations in 5′TC motifs in genomic DNA that contribute to cancer evolution. In this report, we characterized APOBEC1, which is a possible cancer related APOBEC since APOBEC1 mRNA is highly expressed in certain types of tumors, such as lung adenocarcinoma. We found a low level of APOBEC1-induced DNA damage, as measured by γH2AX foci, in genomic DNA of a lung cancer cell line that correlated to its inability to compete in vitro with replication protein A (RPA) for ssDNA. This suggests that RPA can act as a defense against off-target deamination for some APOBEC enzymes. Overall, the data support the model that the ability of an APOBEC to compete with RPA can better predict genomic damage than combined analysis of mRNA expression levels in tumors and analysis of mutation signatures.

Sign in / Sign up

Export Citation Format

Share Document