Requirements for the insertion of the Sindbis envelope glycoproteins into the endoplasmic reticulum membrane

Previous work has shown that the Sindbis structural proteins, core, the internal protein, and PE2 and E1, the integral membrane glycoproteins are synthesized as a polyprotein from a 26S mRNA; core PE2 and E1 are derived by proteolytic cleavage of a nascent chain. Newly synthesized core protein remains on the cytoplasmic side of the endoplasmic reticulum while newly synthesized PE2 and E1 are inserted into the lipid bilayer, presumably via their amino-termini. PE2 and E1 are glycosylated as nascent chains. Here, we examine a temperature-sensitive mutant of Sindbis virus which fails to cleave the structural proteins, resulting in the production of a polyprotein of 130,000 mol wt in which the amino-termini of PE2 and E1 are internal to the protein. Although the envelope sequences are present in this protein, it is not inserted into the endoplasmic reticulum bilayer, but remains on the cytoplasmic side as does the core protein in cells infected with wild-type Sindbis virus. We have also examined the fate of PE2 and E1 in cells treated with tunicamycin, an inhibitor of glycosylation. Unglycosylated PE2 and E1 are inserted normally into the lipid bilayer as are the glycosylated proteins. These results are consistent with the notion that a specific amino-terminal sequence is required for the proper insertion of membrane proteins into the endoplasmic reticulum bilayer, but that glycosylation is not required for this insertion.     

Structure of the murine leukemia virus envelope glycoprotein precursor.

The glycosylated env gene precurosr (Pr80env) of Moloney murine leukemia virus has been isolated by selective immunoprecipitation. Use of the drug tunicamycin to inhibit nascent glycosylation or specific cleavage with endoglycosidase H demonstrated that the precursor contained an apoprotein with a molecular weight of 60,000. The finished virion glycoprotein (gp70) was largely resistant to the action of endoglycosidase H. Chromatography of the glycopeptides of Pr80env in conjunction with endoglycosidase H digestion studies suggested that the precursor contained two distinct major glycosylation sites. Analysis of partial proteolytic cleavage fragments of Pr80env before and after endoglycosidase H treatment placed the two glycosylation sites within a 30,000-dalton region of the apoprotein sequence. Kinetic experiments showed that carbohydrate processing as well as proteolytic cleavage are late steps in the maturation of Pr80env.     

Appendix: Synthesis of N-acylglutathione derivatives by dimethylaminopyridine catalysis

A general procedure, involving 4-dimethylaminopyridine-catalysed acylation of the amino groups in the appropriate S-blocked glutathione, is reported for the preparation of N-blocked derivatives of glutathiones.     

Separation and Purification of Antioxidant Peptides from Enzymatically Prepared Scorpion (Buthus martensii Karsch) Protein Hydrolysates

International Journal of Peptide Research and Therapeutics - The purpose of this study was to separate and purify antioxidant peptides from the scorpion (Buthus martensii Karsch) protein...     

Three proline rotamases involved in calcium homeostasis play differential roles in stress tolerance,virulence and calcineurin regulation of Beauveria bassiana

FK506‐sensitive proline rotamases (FPRs), also known as FK506‐binding proteins (FKBPs), can mediate immunosuppressive drug resistance in budding yeast but their physiological roles in filamentous fungi remain opaque. Here, we report that three FPRs (cytosolic/nuclear 12.15‐kD Fpr1, membrane‐associated 14.78‐kD Fpr2 and nuclear 50.43‐kD Fpr3) are all equally essential for cellular Ca²⁺ homeostasis and contribute significantly to calcineurin activity at different levels in the insect‐pathogenic fungus Beauveria bassiana although the deletion of fpr1 alone conferred resistance to FK506. Radial growth, conidiation, conidial viability and virulence were less compromised in the absence of fpr1 or fpr2 than in the absence of fpr3, which abolished almost all growth on scant media and reduced growth moderately on rich media. The Δfpr3 mutant was more sensitive to Na⁺, K⁺, Mn²⁺, Ca²⁺, Cu²⁺, metal chelate, heat shock and UVB irradiation than was Δfpr2 while both mutants were equally sensitive to Zn²⁺, Mg²⁺, Fe²⁺, H₂O₂ and cell wall‐perturbing agents. In contrast, the Δfpr1 mutant was less sensitive to fewer stress cues. Most of 32 examined genes involved in DNA damage repair, Na⁺/K⁺ detoxification or osmotolerance and Ca²⁺ homeostasis were downregulated sharply in Δfpr2 and Δfpr3 but rarely so affected in Δfpr1, coinciding well with their phenotypic changes. These findings uncover important, but differential, roles of three FPRs in the fungal adaptation to insect host and environment and provide novel insight into their essential roles in calcium signalling pathway.     

On the use of adenovirus dodecahedron as a carrier for glycoconjugate vaccines

Glycoconjugate Journal - Virus-Like Particles (VLPs) have been used as immunogenic molecules in numerous recombinant vaccines. VLPs can also serve as vaccine platform to exogenous antigens, usually...