Intramolecular quality control: HIV-1 envelope gp160 signal-peptide cleavage as a functional folding checkpoint.

Removal of the membrane-tethering signal peptides that target secretory proteins to the endoplasmic reticulum is a prerequisite for proper folding. While generally thought to be removed co-translationally, we report two additional post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves folding fidelity by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine. Simultaneously, the signal peptide delays folding by tethering the N terminus to the membrane, until assembly with the C terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine are both highly conserved and important for viral fitness. Considering the ∼15% proteins with signal peptides and the frequency of N-to-C contacts in protein structures, these regulatory roles of signal peptides are bound to be more common in secretory-protein biogenesis.

Structure and topology around the cleavage site regulate post-translational cleavage of the HIV-1 gp160 signal peptide.

Like all other secretory proteins, the HIV-1 envelope glycoprotein gp160 is targeted to the endoplasmic reticulum (ER) by its signal peptide during synthesis. Proper gp160 folding in the ER requires core glycosylation, disulfide-bond formation and proline isomerization. Signal-peptide cleavage occurs only late after gp160 chain termination and is dependent on folding of the soluble subunit gp120 to a near-native conformation. We here detail the mechanism by which co-translational signal-peptide cleavage is prevented. Conserved residues from the signal peptide and residues downstream of the canonical cleavage site form an extended alpha-helix in the ER membrane, which covers the cleavage site, thus preventing cleavage. A point mutation in the signal peptide breaks the alpha helix allowing co-translational cleavage. We demonstrate that postponed cleavage of gp160 enhances functional folding of the molecule. The change to early cleavage results in decreased viral fitness compared to wild-type HIV.

Deletion of the highly conserved N-glycan at Asn260 of HIV-1 gp120 affects folding and lysosomal degradation of gp120, and results in loss of viral infectivity.

N-linked glycans covering the surface of the HIV-1 glycoprotein gp120 are of major importance for the correct folding of this glycoprotein. Of the, on average, 24 N-linked glycans present on gp120, the glycan at Asn260 was reported to be essential for the correct expression of gp120 and gp41 in the virus particle and deletion of the N260 glycan in gp120 heavily compromised virus infectivity. We show here that gp160 containing the N260Q mutation reaches the Golgi apparatus during biosynthesis. Using pulse-chase experiments with [35S] methionine/cysteine, we show that oxidative folding was slightly delayed in case of mutant N260Q gp160 and that CD4 binding was markedly compromised compared to wild-type gp160. In the search of compensatory mutations, we found a mutation in the V1/V2 loop of gp120 (S128N) that could partially restore the infectivity of mutant N260Q gp120 virus. However, the mutation S128N did not enhance any of the above-mentioned processes so its underlying compensatory mechanism must be a conformational effect that does not affect CD4 binding per se. Finally, we show that mutant N260Q gp160 was cleaved to gp120 and gp41 to a much lower extent than wild-type gp160, and that it was subject of lysosomal degradation to a higher extent than wild-type gp160 showing a prominent role of this process in the breakdown of N260-glycan-deleted gp160, which could not be counteracted by the S128N mutation. Moreover, at least part of the wild-type or mutant gp160 that is normally targeted for lysosomal degradation reached a conformation that enabled CD4 binding.

Carbon dioxide does not affect the methylation status of prognostic important oncogenes Rassf1A and DCR2 in neuroblastoma cells.

PURPOSE: The aim of the current study was to investigate effects of CO(2) atmosphere, mimicking conditions of the pneumoperitoneum during laparoscopy, on epigenetic conditions of Rassf1A and DCR2 oncogenes in neuroblastoma cells. METHODS: SH-SY5Y neuroblastoma cells were exposed to 100% CO(2) for 4 h. Cells were lysed 4, 8 and 168 h after exposure. After methylation analysis of Rassf1A and DCR2 with polymerase chain reaction, results were compared to those of physiologically incubated neuroblastoma cells. RESULTS: No significant changes were found after exposure to carbon dioxide compared to the control. Values of methylated Rassf1A were 12.6 +/- 1.1 versus 13.2 +/- 1.4 ng/microl in the controls, respectively (4 h after incubation), 12.6 +/- 1.2 versus 15.1 +/- 0.9 ng/microl (8 h) and 14.2 +/- 1.5 versus 11.7 +/- 1.3 ng/microl (168 h). DCR2 showed values of 4.6 +/- 0.5 versus 3.7 +/- 0.5 ng/microl (4 h), 3.8 +/- 0.5 versus 4.1 +/- 0.4 ng/microl (8 h) and 3.6 +/- 0.4 versus 3.8 +/- 0.5 ng/microl (168 h). CONCLUSION: Exposure of neuroblastoma cells to 100% CO(2) does not alter methylation of two prognostic relevant index genes. It seems therefore unlikely that effects on methylation levels within CO(2) pneumoperitoneum lead to epigenetic changes in neuroblastoma.