Protein secretion in eukaryotes and prokaryotes involves a universally conserved protein translocation channel formed by the Sec61 complex. Unrelated small-molecule natural products and synthetic ...compounds inhibit Sec61 with differential effects for different substrates or for Sec61 from different organisms, making this a promising target for therapeutic intervention. To understand the mode of inhibition and provide insight into the molecular mechanism of this dynamic translocon, we determined the structure of mammalian Sec61 inhibited by the Mycobacterium ulcerans exotoxin mycolactone via electron cryo-microscopy. Unexpectedly, the conformation of inhibited Sec61 is optimal for substrate engagement, with mycolactone wedging open the cytosolic side of the lateral gate. The inability of mycolactone-inhibited Sec61 to effectively transport substrate proteins implies that signal peptides and transmembrane domains pass through the site occupied by mycolactone. This provides a foundation for understanding the molecular mechanism of Sec61 inhibitors and reveals novel features of translocon function and dynamics.
Display omitted
•The inhibited Sec translocon adopts a conformation optimal for substrate engagement•The inhibitor mycolactone wedges open the lateral gate of Sec61α•Mycolactone blocks the path taken by the signal peptide during engagement•Resistance mutations are likely to operate by modulating translocon dynamics
Gérard et al. determine the structure of the mammalian Sec translocon in an inhibited state. Mycolactone holds the translocon in a conformation optimal for substrate engagement, wedging open the cytosolic side of the lateral gate, while also blocking the path taken by the signal peptide during engagement.
Although antigen cross-presentation in dendritic cells (DCs) is critical to the initiation of most cytotoxic immune responses, the intracellular mechanisms and traffic pathways involved are still ...unclear. One of the most critical steps in this process, the export of internalized antigen to the cytosol, has been suggested to be mediated by Sec61. Sec61 is the channel that translocates signal peptide-bearing nascent polypeptides into the endoplasmic reticulum (ER), and it was also proposed to mediate protein retrotranslocation during ER-associated degradation (a process called ERAD). Here, we used a newly identified Sec61 blocker, mycolactone, to analyze Sec61's contribution to antigen cross-presentation, ERAD, and transport of internalized antigens into the cytosol. As shown previously in other cell types, mycolactone prevented protein import into the ER of DCs. Mycolactone-mediated Sec61 blockade also potently suppressed both antigen cross-presentation and direct presentation of synthetic peptides to CD8
T cells. In contrast, it did not affect protein export from the ER lumen or from endosomes into the cytosol, suggesting that the inhibition of cross-presentation was not related to either of these trafficking pathways. Proteomic profiling of mycolactone-exposed DCs showed that expression of mediators of antigen presentation, including MHC class I and β2 microglobulin, were highly susceptible to mycolactone treatment, indicating that Sec61 blockade affects antigen cross-presentation indirectly. Together, our data suggest that the defective translocation and subsequent degradation of Sec61 substrates is the cause of altered antigen cross-presentation in Sec61-blocked DCs.
Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this ...toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen2
that improves the ratio of complemented signal to the background of FP
-expressing cells compared to the commonly used split GFP
; as well as a 10-fold brighter red-colored split-sfCherry2
. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry2
and GFP
, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use.
Selenium is a fascinating element that has a long history, most of which documents it as a deleterious element to health. In more recent years, selenium has been found to be an essential element in ...the diet of humans, all other mammals, and many other life forms. It has many health benefits that include, for example, roles in preventing heart disease and certain forms of cancer, slowing AIDS progression in HIV patients, supporting male reproduction, inhibiting viral expression, and boosting the immune system, and it also plays essential roles in mammalian development. Elucidating the molecular biology of selenium over the past 40 years generated an entirely new field of science which encompassed the many novel features of selenium. These features were (1) how this element makes its way into protein as the 21st amino acid in the genetic code, selenocysteine (Sec); (2) the vast amount of machinery dedicated to synthesizing Sec uniquely on its tRNA; (3) the incorporation of Sec into protein; and (4) the roles of the resulting Sec-containing proteins (selenoproteins) in health and development. One of the research areas receiving the most attention regarding selenium in health has been its role in cancer prevention, but further research has also exposed the role of this element as a facilitator of various maladies, including cancer.
This paper comments on the some mistakes which have been observed in Tripathi et al. (2023) 1. We have specifically pointed out the following mistakes of Tripathi et al. (2023) 1.
i) Presented (41, ...32) SEC-DAEC-TAEC H-matrix in Fig. 3 of Tripathi et al. (2023) 1.
ii) Presented error detection and correction coverage in Table 6 of Tripathi et al. (2023) 1.
The corrected versions of the above mentioned mistakes have been presented in this paper. Also the FPGA-based synthesis result of corrected encoder and decoder circuits based on the corrected (41, 32) H-matrix have been presented here.
The endoplasmic reticulum (ER) is a nexus for mRNA localization and translation, and recent studies have demonstrated that ER-bound ribosomes also play a transcriptome-wide role in regulating ...proteome composition. The Sec61 translocon (SEC61) serves as the receptor for ribosomes that translate secretory/integral membrane protein-encoding mRNAs, but whether SEC61 also serves as a translation site for cytosolic protein-encoding mRNAs remains unknown. Here, using a BioID proximity-labeling approach in HEK293T Flp-In cell lines, we examined interactions between ER-resident proteins and ribosomes in vivo. Using in vitro analyses, we further focused on bona fide ribosome interactors (i.e. SEC61) and ER proteins (ribophorin I, leucine-rich repeat–containing 59 (LRRC59), and SEC62) previously implicated in associating with ribosomes. We observed labeling of ER-bound ribosomes with the SEC61β and LRRC59 BioID reporters, comparatively modest labeling with the ribophorin I reporter, and no labeling with the SEC62 reporter. A biotin pulse-chase/subcellular fractionation approach to examine ribosome exchange at the SEC61β and LRRC59 sites revealed that, at steady state, ribosomes at these sites comprise both rapid- and slow-exchanging pools. Global translational initiation arrest elicited by the inhibitor harringtonine accelerated SEC61β reporter-labeled ribosome exchange. RNA-Seq analyses of the mRNAs associated with SEC61β- and LRRC59-labeled ribosomes revealed both site-enriched and shared mRNAs and further established that the ER has a transcriptome-wide role in regulating proteome composition. These results provide evidence that ribosomes interact with the ER membrane via multiple modes and suggest regulatory mechanisms that control global proteome composition via ER membrane-bound ribosomes.
•We examine the effect of MMoU adoption on firms’ debt choice.•Countries’ MMoU adoption increases firms’ reliance on public debt.•Improved SEC oversight of firms post-MMoU is a potential channel for ...this effect.•We contribute to the literature on the economic implications of the MMoU.
The objective of this study is to examine the role of regulatory oversight in firms’ choice of debt. Using the signing of the Multilateral Memorandum of Understanding (MMoU) as a quasi-natural experiment, we find that enhanced SEC oversight facilitates public debt contracting, especially for firms with severe agency problems. Our study has important implications for both research and practice.
Translation regulation of mammalian selenoproteins Vindry, Caroline; Ohlmann, Théophile; Chavatte, Laurent
Biochimica et biophysica acta. General subjects,
11/2018, Letnik:
1862, Številka:
11
Journal Article
Recenzirano
Interest in selenium research has considerably grown over the last decades owing to the association of selenium deficiencies with an increased risk of several human diseases, including cancers, ...cardiovascular disorders and infectious diseases. The discovery of a genetically encoded 21st amino acid, selenocysteine, is a fascinating breakthrough in molecular biology as it is the first addition to the genetic code deciphered in the 1960s. Selenocysteine is a structural and functional analog of cysteine, where selenium replaces sulfur, and its presence is critical for the catalytic activity of selenoproteins.
The insertion of selenocysteine is a non-canonical translational event, based on the recoding of a UGA codon in selenoprotein mRNAs, normally used as a stop codon in other cellular mRNAs. Two RNA molecules and associated partners are crucial components of the selenocysteine insertion machinery, the Sec-tRNASerSec devoted to UGA codon recognition and the SECIS elements located in the 3′UTR of selenoprotein mRNAs.
The translational UGA recoding event is a limiting stage of selenoprotein expression and its efficiency is regulated by several factors.
The control of selenoproteome expression is crucial for redox homeostasis and antioxidant defense of mammalian organisms. In this review, we summarize current knowledge on the co-translational insertion of selenocysteine into selenoproteins, and its layers of regulation.
Display omitted
•Insertion of selenocysteine in selenoproteins is a non-canonical translational event, based on the recoding of a UGA codon, normally a stop codon•The Sec-tRNASerSec, the SECIS element and associated partners are central components for selenocysteine insertion•The translational UGA recoding is a limiting stage that controls selenoprotein expression in response to various stimuli
Many proteins must translocate through the protein-conducting Sec61 channel in the eukaryotic endoplasmic reticulum membrane or the SecY channel in the prokaryotic plasma membrane
. Proteins with ...highly hydrophobic signal sequences are first recognized by the signal recognition particle (SRP)
and then moved co-translationally through the Sec61 or SecY channel by the associated translating ribosome. Substrates with less hydrophobic signal sequences bypass the SRP and are moved through the channel post-translationally
. In eukaryotic cells, post-translational translocation is mediated by the association of the Sec61 channel with another membrane protein complex, the Sec62-Sec63 complex
, and substrates are moved through the channel by the luminal BiP ATPase
. How the Sec62-Sec63 complex activates the Sec61 channel for post-translational translocation is not known. Here we report the electron cryo-microscopy structure of the Sec complex from Saccharomyces cerevisiae, consisting of the Sec61 channel and the Sec62, Sec63, Sec71 and Sec72 proteins. Sec63 causes wide opening of the lateral gate of the Sec61 channel, priming it for the passage of low-hydrophobicity signal sequences into the lipid phase, without displacing the channel's plug domain. Lateral channel opening is triggered by Sec63 interacting both with cytosolic loops in the C-terminal half of Sec61 and transmembrane segments in the N-terminal half of the Sec61 channel. The cytosolic Brl domain of Sec63 blocks ribosome binding to the channel and recruits Sec71 and Sec72, positioning them for the capture of polypeptides associated with cytosolic Hsp70
. Our structure shows how the Sec61 channel is activated for post-translational protein translocation.
Described here is the current status of the upgraded in situ size‐exclusion chromatography (SEC) system implemented with the D22 small‐angle neutron scattering (SANS) instrument at the Institut ...Laue–Langevin. Since its initial proof of principle in 2016, this SEC–SANS arrangement has been continuously requested by the user community, leading to the design of an upgraded version. A detailed description of the setup and its control is provided, and a few examples of protein structural investigations are presented, which will highlight the various possibilities and limitations of the setup to optimize experimental success.
This publication describes the stable version of a liquid chromatography system combined with the small‐angle neutron scattering (SANS) instrument D22 at Institut Laue–Langevin to run size‐exclusion chromatography (SEC) immediately before SANS measurement.
PDF
