scholarly journals Intramolecular quality control: HIV-1 Envelope gp160 signal-peptide cleavage as a functional folding checkpoint

2020 ◽  
Author(s):  
Nicholas McCaul ◽  
Matthias Quandte ◽  
Ilja Bontjer ◽  
Guus van Zadelhoff ◽  
Aafke Land ◽  
...  
Keyword(s):  
Quality Control ◽  
Signal Peptide ◽  
Protein Biosynthesis ◽  
Protein Structures ◽  
Secretory Protein ◽  
Viral Fitness ◽  
Secretory Proteins ◽  
Redox Active ◽  
Hiv 1 ◽  
Redox Active Cysteine

SummaryRemoval 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 well before translation termination, we here report two novel post-targeting functions for the HIV-1 gp120 signal peptide, which remains attached until gp120 folding triggers its removal. First, the signal peptide improves fidelity of folding by enhancing conformational plasticity of gp120 by driving disulfide isomerization through a redox-active cysteine, at the same time delaying folding by tethering the N-terminus to the membrane, which needs assembly with the C-terminus. Second, its carefully timed cleavage represents intramolecular quality control and ensures release and stabilization of (only) natively folded gp120. Postponed cleavage and the redox-active cysteine both are highly conserved and important for viral fitness. Considering the ∼15% secretory proteins in our genome and the frequency of N-to-C contacts in protein structures, these regulatory roles of the signal peptide are bound to be more common in secretory-protein biosynthesis.

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

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Erik Lee Snapp ◽  
Nicholas McCaul ◽  
Matthias Quandte ◽  
Zuzana Cabartova ◽  
Ilja Bontjer ◽  
...  
Keyword(s):  
Signal Peptide ◽  
Cleavage Site ◽  
Alpha Helix ◽  
Viral Fitness ◽  
Secretory Proteins ◽  
Early Cleavage ◽  
Conserved Residues ◽  
Peptide Cleavage ◽  
Proline Isomerization ◽  
Hiv 1

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.

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

Cell Reports ◽  
2021 ◽  
Vol 36 (9) ◽  
pp. 109646
Author(s):  
Nicholas McCaul ◽  
Matthias Quandte ◽  
Ilja Bontjer ◽  
Guus van Zadelhoff ◽  
Aafke Land ◽  
...  
Keyword(s):  
Quality Control ◽  
Signal Peptide ◽  
Peptide Cleavage ◽  
Hiv 1

Protein Quality Control of the Endoplasmic Reticulum and Ubiquitin–Proteasome-Triggered Degradation of Aberrant Proteins: Yeast Pioneers the Path

2018 ◽  
Vol 87 (1) ◽  
pp. 751-782 ◽  
Author(s):  
Nicole Berner ◽  
Karl-Richard Reutter ◽  
Dieter H. Wolf
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Protein Quality ◽  
Protein Structures ◽  
Protein Quality Control ◽  
Ubiquitin Proteasome System ◽  
Misfolded Proteins ◽  
Secretory Proteins ◽  
Structural Alterations ◽  
Ubiquitin Proteasome

Cells must constantly monitor the integrity of their macromolecular constituents. Proteins are the most versatile class of macromolecules but are sensitive to structural alterations. Misfolded or otherwise aberrant protein structures lead to dysfunction and finally aggregation. Their presence is linked to aging and a plethora of severe human diseases. Thus, misfolded proteins have to be rapidly eliminated. Secretory proteins constitute more than one-third of the eukaryotic proteome. They are imported into the endoplasmic reticulum (ER), where they are folded and modified. A highly elaborated machinery controls their folding, recognizes aberrant folding states, and retrotranslocates permanently misfolded proteins from the ER back to the cytosol. In the cytosol, they are degraded by the highly selective ubiquitin–proteasome system. This process of protein quality control followed by proteasomal elimination of the misfolded protein is termed ER-associated degradation (ERAD), and it depends on an intricate interplay between the ER and the cytosol.

Using enrichment index for quality control of secretory protein sample and identification of secretory proteins

2009 ◽  
Vol 44 (3) ◽  
pp. 397-403 ◽  
Author(s):  
Yong Chen ◽  
Bei Gu ◽  
Shuzhen Wu ◽  
Wei Sun ◽  
Sucan Ma ◽  
...  
Keyword(s):  
Quality Control ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Protein Sample ◽  
Enrichment Index

scholarly journals Export of a Cysteine-Free Misfolded Secretory Protein from the Endoplasmic Reticulum for Degradation Requires Interaction with Protein Disulfide Isomerase

1999 ◽  
Vol 147 (7) ◽  
pp. 1443-1456 ◽  
Author(s):  
Pauline Gillece ◽  
José Manuel Luz ◽  
William J. Lennarz ◽  
Francisco Javier de la Cruz ◽  
Karin Römisch
Keyword(s):  
Quality Control ◽  
Protein Disulfide Isomerase ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Disulfide Isomerase ◽  
Thiol Content ◽  
Mutant Forms ◽  
Protein Disulfide ◽  
Chemical Cross Linking ◽  
Er Membrane

Protein disulfide isomerase (PDI) interacts with secretory proteins, irrespective of their thiol content, late during translocation into the ER; thus, PDI may be part of the quality control machinery in the ER. We used yeast pdi1 mutants with deletions in the putative peptide binding region of the molecule to investigate its role in the recognition of misfolded secretory proteins in the ER and their export to the cytosol for degradation. Our pdi1 deletion mutants are deficient in the export of a misfolded cysteine-free secretory protein across the ER membrane to the cytosol for degradation, but ER-to-Golgi complex transport of properly folded secretory proteins is only marginally affected. We demonstrate by chemical cross-linking that PDI specifically interacts with the misfolded secretory protein and that mutant forms of PDI have a lower affinity for this protein. In the ER of the pdi1 mutants, a higher proportion of the misfolded secretory protein remains associated with BiP, and in export-deficient sec61 mutants, the misfolded secretory protein remain bounds to PDI. We conclude that the chaperone PDI is part of the quality control machinery in the ER that recognizes terminally misfolded secretory proteins and targets them to the export channel in the ER membrane.

scholarly journals Size-Dependent Secretory Protein Reflux into the Cytosol in Association with Acute Endoplasmic Reticulum Stress

2019 ◽  
Author(s):  
Patrick Lajoie ◽  
Erik L. Snapp
Keyword(s):  
Quality Control ◽  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Fluorescent Protein ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Dependent Manner ◽  
Size Dependent ◽  
Heat Shock Stress ◽  
Shock Stress

ABSTRACTOnce secretory proteins have been targeted to the endoplasmic reticulum (ER), the proteins typically remain partitioned from the cytosol. If the secretory proteins misfold, they can be unfolded and retrotranslocated into the cytosol for destruction by the proteasome by ER-associated protein Degradation (ERAD). Here, we report that correctly folded and targeted luminal ER fluorescent protein reporters accumulate in the cytosol during acute misfolded secretory protein stress in yeast. Photoactivation fluorescence microscopy experiments reveal that luminal reporters already localized to the ER relocalize to the cytosol, even in the absence of essential ERAD machinery. We named this process “ER reflux.” Reflux appears to be regulated in a size-dependent manner for reporters. Interestingly, prior heat shock stress also prevents ER stress-induced reflux. Together, our findings establish a new ER stress-regulated pathway for relocalization of small luminal secretory proteins into the cytosol, distinct from the ERAD and pre-emptive quality control pathways.

scholarly journals Identification of signal sequence binding proteins integrated into the rough endoplasmic reticulum membrane

1987 ◽  
Vol 242 (3) ◽  
pp. 767-777 ◽  
Author(s):  
A Robinson ◽  
M A Kaderbhai ◽  
B M Austen
Keyword(s):  
Endoplasmic Reticulum ◽  
Rough Endoplasmic Reticulum ◽  
Signal Peptide ◽  
Signal Sequence ◽  
Secretory Protein ◽  
Phospholipid Bilayer ◽  
Signal Peptidase ◽  
Secretory Proteins ◽  
Endoplasmic Reticulum Membrane

An azidophenacyl derivative of a chemically synthesized consensus signal peptide has been prepared. The peptide, when photoactivated in the presence of rough or high-salt-stripped microsomes from pancreas, leads to inhibition of their activity in cotranslational processing of secretory pre-proteins translated from their mRNA in vitro. The peptide binds specifically with high affinity to components in the microsomal membranes from pancreas and liver, and photoreaction of a radioactive form of the azidophenacyl derivative leads to covalent linkage to yield two closely related radiolabelled proteins of Mr about 45,000. These proteins are integrated into the membrane, with large 30,000-Mr domains embedded into the phospholipid bilayer to which the signal peptide binds. A smaller, endopeptidase-sensitive, domain is exposed on the cytoplasmic surface of the microsomal vesicles. The specificity and selectivity of the binding of azidophenacyl-derivatized consensus signal peptide was demonstrated by concentration-dependent inhibition of photolabelling by the ‘cold’ synthetic consensus signal peptide and by a natural internal signal sequence cleaved and isolated from ovalbumin. The properties of the labelled 45,000-Mr protein-signal peptide complexes, i.e. mass, pI, ease of dissociation from the membrane by detergent or salts and immunological properties, distinguish them from other proteins, e.g. subunits of signal recognition particle, docking protein and signal peptidase, already known to be involved in targetting and processing of nascent secretory proteins at the rough endoplasmic reticulum membrane. Although the 45,000-Mr signal peptide binding protein displays properties similar to those of the signal peptidase, a component of the endoplasmic reticulum, the azido-derivatized consensus signal peptide does not interact with it. It is proposed that the endoplasmic reticulum proteins with which the azidophenacyl-derivatized consensus signal peptide interacts to yield the 45,000-Mr adducts may act as receptors for signals in nascent secretory pre-proteins in transduction of changes in the endoplasmic reticulum which bring about translocation of secretory protein across the membrane.

Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system

2013 ◽  
Vol 8 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Yasuhiko Soejima ◽  
Jae Lee ◽  
Yudai Nagata ◽  
Hiroaki Mon ◽  
Kazuhiro Iiyama ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Signal Peptide ◽  
Protein Production ◽  
Expression System ◽  
Secretory Protein ◽  
Secretory Proteins ◽  
Signal Peptides ◽  
Secretion Pathway ◽  
Key Factor ◽  
Secretion Efficiency

AbstractThe baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.

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