Hypoaldosteronism due to a novel SEC61A1 variant successfully treated with fludrocortisone.

Background: Genetic variants in SEC61A1 are associated with autosomal dominant tubulointerstitial kidney disease. SEC61A1 is a translocon in the endoplasmic reticulum membrane and variants affect biosynthesis of renin and uromodulin. Methods: A patient is described that presented at 1 year of age with failure-to-thrive, kidney failure (glomerular filtration rate, GFR, 18 ml/min/1.73m2), hyperkalemia and acidosis. Genetic evaluation was performed by whole genome sequencing. Results: The patient has a novel de novo heterozygous SEC61A1 variant, Phe458Val. Plasma renin was low or normal, aldosterone was low or undetectable and uromodulin was low. Kidney biopsy at 2 years exhibited subtle changes suggestive of tubular dysgenesis without tubulocystic or glomerulocystic lesions and with renin staining of the juxtaglomerular cells. The patient experienced extreme fatigue due to severe hypotension attributed to hypoaldosteronism and at 8 years of age fludrocortisone treatment was initiated with marked improvement in her well-being. Blood pressure and potassium normalized. Biopsy at 9 years showed extensive glomerulosclerosis and mild tubulointerstitial fibrosis, as well as tubular mitochondrial abnormalities, without specific diagnostic changes. Her GFR improved to 54 ml/min/1.73m2. Conclusions: As the renin-angiotensin system promotes aldosterone release, and the patient had repeatedly undetectable aldosterone levels, the SEC61A1 variant presumably contributed to severe hypotension. Treatment with a mineralocorticoid had a beneficial effect and corrected the electrolyte and acid-base disorder. We suggest that the increased blood pressure hemodynamically improved the patient's kidney function.

Inter-compatibility of eukaryotic and Asgard archaea ribosome-translocon machineries.

In all domains of life, the ribosome-translocon complex inserts nascent transmembrane proteins into, and processes and transports signal peptide-containing proteins across, membranes. Eukaryotic translocons are anchored in the endoplasmic reticulum, while the prokaryotic complexes reside in cell membranes. Phylogenetic analyses indicate inheritance of eukaryotic Sec61/OST/TRAP translocon subunits from an Asgard archaea ancestor. However, the mechanism for translocon migration from a peripheral membrane to an internal cellular compartment (the proto-endoplasmic reticulum) during eukaryogenesis is unknown. Here we show compatibility between the eukaryotic ribosome-translocon complex and Asgard signal peptides and transmembrane proteins. We find that Asgard translocon proteins from Candidatus Prometheoarchaeum syntrophicum strain MK-D1, a Lokiarchaeon confirmed to contain no internal cellular membranes, are targeted to the eukaryotic endoplasmic reticulum on ectopic expression. Furthermore, we show that the cytoplasmic domain of MK-D1 OST1 (ribophorin I) can interact with eukaryotic ribosomes. Our data indicate that the location of existing ribosome-translocon complexes, at the protein level, determines the future placement of yet to be translated translocon subunits. This principle predicts that during eukaryogenesis, under positive selection pressure, the relocation of a few translocon complexes to the proto-endoplasmic reticulum will have contributed to propagating the new translocon location, leading to their loss from the cell membrane.

SEC61 translocon gamma subunit is correlated with glycolytic activity, epithelial mesenchymal transition and the immune suppressive phenotype of lung adenocarcinoma.

Lung adenocarcinoma (LUAD) remains a predominant cause of cancer-related mortality globally, underscoring the urgency for targeted therapeutic strategies. The specific role and impact of the SEC61 translocon gamma subunit (SEC61G) in LUAD progression and metastasis remain largely unexplored. In this study, we use a multifaceted approach, combining bioinformatics analysis with experimental validation, to elucidate the pivotal role of SEC61G and its associated molecular mechanisms in LUAD. Our integrated analyses reveal a significant positive correlation between SEC61G expression and the glycolytic activity of LUAD, as evidenced by increased fluorodeoxyglucose (FDG) uptake on positron emission tomography (PET)/CT scans. Further investigations show the potential influence of SEC61G on metabolic reprogramming, which contributes to the immunosuppressive tumor microenvironment (TME). Remarkably, we identify a negative association between SEC61G expression levels and the infiltration of critical immune cell populations within the TME, along with correlations with immune checkpoint gene expression and tumor heterogeneity scores in LUAD. Functional studies demonstrate that SEC61G knockdown markedly inhibits the migration of A549 and H2030 LUAD cells. This inhibitory effect is accompanied by a significant downregulation of key regulators of tumor progression, including hypoxia-inducible factor-1 alpha (HIF-1α), lactate dehydrogenase A, and genes involved in the epithelial-mesenchymal transition pathway. In conclusion, our comprehensive analyses position SEC61G as a potential prognostic biomarker intricately linked to glycolytic metabolism, the EMT pathway, and the establishment of an immune-suppressive phenotype in LUAD. These findings underscore the potential of SEC61G as a therapeutic target and predictive marker for immunotherapeutic responses in LUAD patients.

The RNA Helicase Ded1 from Yeast Is Associated with the Signal Recognition Particle and Is Regulated by SRP21.

The DEAD-box RNA helicase Ded1 is an essential yeast protein involved in translation initiation that belongs to the DDX3 subfamily. The purified Ded1 protein is an ATP-dependent RNA-binding protein and an RNA-dependent ATPase, but it was previously found to lack substrate specificity and enzymatic regulation. Here we demonstrate through yeast genetics, yeast extract pull-down experiments, in situ localization, and in vitro biochemical approaches that Ded1 is associated with, and regulated by, the signal recognition particle (SRP), which is a universally conserved ribonucleoprotein complex required for the co-translational translocation of polypeptides into the endoplasmic reticulum lumen and membrane. Ded1 is physically associated with SRP components in vivo and in vitro. Ded1 is genetically linked with SRP proteins. Finally, the enzymatic activity of Ded1 is inhibited by SRP21 in the presence of SCR1 RNA. We propose a model where Ded1 actively participates in the translocation of proteins during translation. Our results provide a new understanding of the role of Ded1 during translation.

RAMPing up knowledge of the translocon.

Advanced cryo-EM approaches reveal surprising insights into the molecular structure that allows nascent proteins to be inserted into the membrane of the endoplasmic reticulum.

High SEC61A1 expression predicts poor outcome of acute myeloid leukemia.

The malfunction of SEC61A1 has been linked to several types of cancers, but its role in acute myeloid leukemia (AML) remains poorly understood. In this study, we used a series of bioinformatics analysis techniques, including gene expression profiling and proteomic analysis. Our findings were subsequently validated through a series of in vitro experiments, such as SEC61A1 knockdown in cell lines and RT-qPCR. We discovered a significant up-regulation of SEC61A1 in AML patients compared to healthy controls. AML patients with elevated SEC61A1 expression exhibited reduced overall survival compared to those with lower expression. Moreover, SEC61A1 expression emerged as an independent risk factor for predicting the survival of AML patients undergoing allo-HSCT. Our analysis also revealed an association between high SEC61A1 expression and increased signaling pathways related to cell growth. Our study underscores the importance of SEC61A1 expression as a novel prognostic indicator for predicting survival among AML patients, while also identifying it as a promising therapeutic target.

Brucella-driven host N-glycome remodeling controls infection.

Many powerful methods have been employed to elucidate the global transcriptomic, proteomic, or metabolic responses to pathogen-infected host cells. However, the host glycome responses to bacterial infection remain largely unexplored, and hence, our understanding of the molecular mechanisms by which bacterial pathogens manipulate the host glycome to favor infection remains incomplete. Here, we address this gap by performing a systematic analysis of the host glycome during infection by the bacterial pathogen Brucella spp. that cause brucellosis. We discover, surprisingly, that a Brucella effector protein (EP) Rhg1 induces global reprogramming of the host cell N-glycome by interacting with components of the oligosaccharide transferase complex that controls N-linked protein glycosylation, and Rhg1 regulates Brucella replication and tissue colonization in a mouse model of brucellosis, demonstrating that Brucella exploits the EP Rhg1 to reprogram the host N-glycome and promote bacterial intracellular parasitism, thereby providing a paradigm for bacterial control of host cell infection.

2 in 1 Vectors Improve in Planta BiFC and FRET Analysis.

Protein-protein interactions (PPIs) play vital roles in all subcellular processes, and a number of tools have been developed for their detection and analysis. Each method has its unique set of benefits and drawbacks that need to be considered prior application. In fact, researchers are spoilt for choice when it comes to deciding which method to use for the initial detection of a PPI and which to corroborate the findings. With constant improvements in microscope development, the possibilities of techniques to study PPIs in vivo, and in real time, are continuously enhanced and expanded. Here, we describe three common approaches, their recent improvements incorporating a 2-in-1 cloning approach, and their application in plant cell biology: ratiometric bimolecular fluorescence complementation (rBiFC), FRET acceptor photobleaching (FRET-AB), and fluorescent lifetime imaging (FRET-FLIM), using Nicotiana benthamiana leaves and Arabidopsis thaliana cell culture protoplasts as transient expression systems.

Cotranslational sorting and processing of newly synthesized proteins in eukaryotes.

Ribosomes interact with a variety of different protein biogenesis factors that guide newly synthesized proteins to their native 3D shapes and cellular localization. Depending on the type of translated substrate, a distinct set of cotranslational factors must interact with the ribosome in a timely and coordinated manner to ensure proper protein biogenesis. While cytonuclear proteins require cotranslational maturation and folding factors, secretory proteins must be maintained in an unfolded state and processed cotranslationally by transport and membrane translocation factors. Here we explore the specific cotranslational processing steps for cytonuclear, secretory, and membrane proteins in eukaryotes and then discuss how the nascent polypeptide-associated complex (NAC) cotranslationally sorts these proteins into the correct protein biogenesis pathway.

Identification of SEC61G as a Diagnostic and Prognostic Biomarker in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is a heterogeneous malignancy originating from the oral mucosal epithelium. Detecting novel biomarkers can offer crucial information on disease aggressiveness and expected clinical outcomes for individual patients. SEC61G, an aberrantly expressed gene in various cancers, has been associated with negative clinical outcomes. However, its expression and clinical significance in OSCC is still unclear. In the present study, we investigated the SEC61G expression level in OSCC using bioinformatic and immunohistochemical analyses. Additionally, our findings revealed a significant correlation between SEC61G expression and clinicopathological characteristics, as well as a worse prognosis in OSCC patients. Notably, flow cytometry analysis on patient samples revealed that SEC61G expression was also linked to decreased immune infiltration in OSCC patients. In conclusion, our study provides evidence supporting SEC61G's role as a potential diagnostic, prognostic, and therapeutic marker in OSCC.

SEC61 translocon subunit gamma enhances low-dose cisplatin-induced cancer-stem cell properties of head and neck squamous cell carcinoma via enhancing Ca2+-mediated autophagy.

Background/purpose: High SEC61 translocon subunit gamma (SEC61G) expression is associated with an unfavorable prognosis in patients with head and neck squamous cell carcinoma (HNSCC), but the underlying mechanisms remain poorly understood. Materials and methods: HNSCC representative cell lines SCC15 and CAL27 were used to explore the regulation of SEC61G on Ca2+ leak from the endoplasmic reticulum (ER). Ca2+-activated autophagy was monitored by fluorescent labeling of autophagosomes and western blotting assays. CSC marker expression, sphere formation, colony formation, and transwell of invasion were detected to investigate the role of SEC61G in regulating cancer-stem cell (CSC) properties. Results: Among the SEC61 complex genes, only SEC61G upregulation is consistently associated with unfavorable progression-free interval and disease-specific survival in patients with HNSCC. Low-dose cisplatin (CDDP) treatment induced SEC61G upregulation in SCC15 and CAL27 cells. SEC61G knockdown significantly impaired CDDP-induced Ca2+ from the ER and the phosphorylation of ERK1/2 and AMPK. CDDP-induced autophagy in HNSCC cells were hampered by SEC61G shRNA, in terms of impaired autophagosome formation, lowered LC3-II/GAPDH ratio and restored p62 expression. CDDP-induced CSC properties, including CSC marker expression, sphere formation, colony formation, and invasive capabilities could be suppressed by shSEC61G and chloroquine, a specific autophagy inhibitor. Conclusion: Findings of this study revealed the contribution of SEC61G in promoting cisplatin-induced CSC properties of HNSCC cells via promoting Ca2+-mediated autophagy.

Quantitative Mass Spectrometry Characterizes Client Spectra of Components for Targeting of Membrane Proteins to and Their Insertion into the Membrane of the Human ER.

To elucidate the redundancy in the components for the targeting of membrane proteins to the endoplasmic reticulum (ER) and/or their insertion into the ER membrane under physiological conditions, we previously analyzed different human cells by label-free quantitative mass spectrometry. The HeLa and HEK293 cells had been depleted of a certain component by siRNA or CRISPR/Cas9 treatment or were deficient patient fibroblasts and compared to the respective control cells by differential protein abundance analysis. In addition to clients of the SRP and Sec61 complex, we identified membrane protein clients of components of the TRC/GET, SND, and PEX3 pathways for ER targeting, and Sec62, Sec63, TRAM1, and TRAP as putative auxiliary components of the Sec61 complex. Here, a comprehensive evaluation of these previously described differential protein abundance analyses, as well as similar analyses on the Sec61-co-operating EMC and the characteristics of the topogenic sequences of the various membrane protein clients, i.e., the client spectra of the components, are reported. As expected, the analysis characterized membrane protein precursors with cleavable amino-terminal signal peptides or amino-terminal transmembrane helices as predominant clients of SRP, as well as the Sec61 complex, while precursors with more central or even carboxy-terminal ones were found to dominate the client spectra of the SND and TRC/GET pathways for membrane targeting. For membrane protein insertion, the auxiliary Sec61 channel components indeed share the client spectra of the Sec61 complex to a large extent. However, we also detected some unexpected differences, particularly related to EMC, TRAP, and TRAM1. The possible mechanistic implications for membrane protein biogenesis at the human ER are discussed and can be expected to eventually advance our understanding of the mechanisms that are involved in the so-called Sec61-channelopathies, resulting from deficient ER protein import.

Mycolactone A vs. B: Multiscale Simulations Reveal the Roles of Localization and Association in Isomer-Specific Toxicity.

Mycolactone is an exotoxin produced by Mycobacterium ulcerans that causes the neglected tropical skin disease Buruli ulcer. This toxin inhibits the Sec61 translocon in the endoplasmic reticulum (ER), preventing the host cell from producing several secretory and transmembrane proteins, resulting in cytotoxic and immunomodulatory effects. Interestingly, only one of the two dominant isoforms of mycolactone is cytotoxic. Here, we investigate the origin of this specificity by performing extensive molecular dynamics (MD) simulations with enhanced free energy sampling to query the association trends of the two isoforms with both the Sec61 translocon, using two distinct cryo-electron microscopy (cryo-EM) models as references, and the ER membrane, which serves as a toxin reservoir prior to association. Our results suggest that mycolactone B (the cytotoxic isoform) has a stronger association with the ER membrane than mycolactone A due to more favorable interactions with membrane lipids and water molecules. This could increase the reservoir of toxin proximal to the Sec61 translocon. In one model of Sec61 inhibited by mycolactone, we find that isomer B interacts more closely with residues thought to play a key role in signal peptide recognition and, thus, are essential for subsequent protein translocation. In the other model, we find that isomer B interacts more closely with the lumenal and lateral gates of the translocon, the dynamics of which are essential for protein translocation. These interactions induce a more closed conformation, which has been suggested to block signal peptide insertion and subsequent protein translocation. Collectively, these findings suggest that isomer B's unique cytotoxicity is a consequence of both increased localization to the ER membrane and channel-locking association with the Sec61 translocon, facets that could be targeted in the development of Buruli Ulcer diagnostics and Sec61-targeted therapeutics.

SEC61G assists EGFR-amplified glioblastoma to evade immune elimination.

Amplification of chromosome 7p11 (7p11) is the most common alteration in primary glioblastoma (GBM), resulting in gains of epidermal growth factor receptor (EGFR) copy number in 50 to 60% of GBM tumors. However, treatment strategies targeting EGFR have thus far failed in clinical trials, and the underlying mechanism remains largely unclear. We here demonstrate that EGFR amplification at the 7p11 locus frequently encompasses its neighboring genes and identifies SEC61G as a critical regulator facilitating GBM immune evasion and tumor growth. We found that SEC61G is always coamplified with EGFR and is highly expressed in GBM. As an essential subunit of the SEC61 translocon complex, SEC61G promotes translocation of newly translated immune checkpoint ligands (ICLs, including PD-L1, PVR, and PD-L2) into the endoplasmic reticulum and promotes their glycosylation, stabilization, and membrane presentation. Depletion of SEC61G promotes the infiltration and cytolytic activity of CD8+ T cells and thus inhibits GBM occurrence. Further, SEC61G inhibition augments the therapeutic efficiency of EGFR tyrosine kinase inhibitors in mice. Our study demonstrates a critical role of SEC61G in GBM immune evasion, which provides a compelling rationale for combination therapy of EGFR-amplified GBMs.

From Bacterial Toxin to Therapeutic Agent: The Unexpected Fate of Mycolactone.

"Recognizing a surprising fact is the first step towards discovery." This famous quote from Louis Pasteur is particularly appropriate to describe what led us to study mycolactone, a lipid toxin produced by the human pathogen Mycobacterium ulcerans. M. ulcerans is the causative agent of Buruli ulcer, a neglected tropical disease manifesting as chronic, necrotic skin lesions with a "surprising" lack of inflammation and pain. Decades after its first description, mycolactone has become much more than a mycobacterial toxin. This uniquely potent inhibitor of the mammalian translocon (Sec61) helped reveal the central importance of Sec61 activity for immune cell functions, the spread of viral particles and, unexpectedly, the viability of certain cancer cells. We report in this review the main discoveries that marked our research into mycolactone, and the medical perspectives they opened up. The story of mycolactone is not over and the applications of Sec61 inhibition may go well beyond immunomodulation, viral infections, and oncology.

A systematic proximity ligation approach to studying protein-substrate specificity identifies the substrate spectrum of the Ssh1 translocon.

Many cellular functions are carried out by protein pairs or families, providing robustness alongside functional diversity. For such processes, it remains a challenge to map the degree of specificity versus promiscuity. Protein-protein interactions (PPIs) can be used to inform on these matters as they highlight cellular locals, regulation and, in cases where proteins affect other proteins - substrate range. However, methods to systematically study transient PPIs are underutilized. In this study, we create a novel approach to systematically compare stable or transient PPIs between two yeast proteins. Our approach, Cel-lctiv (CELlular biotin-Ligation for Capturing Transient Interactions in vivo), uses high-throughput pairwise proximity biotin ligation for comparing PPIs systematically and in vivo. As a proof of concept, we studied the homologous translocation pores Sec61 and Ssh1. We show how Cel-lctiv can uncover the unique substrate range for each translocon allowing us to pinpoint a specificity determinator driving interaction preference. More generally, this demonstrates how Cel-lctiv can provide direct information on substrate specificity even for highly homologous proteins.

Sec61 blockade therapy overrides resistance to proteasome inhibitors and immunomodulatory drugs in multiple myeloma.

Multiple Myeloma (MM) is an incurable neoplasm of mature B cells and the second most prevalent hematological malignancy worldwide. While combinations of proteasome inhibitors like bortezomib (Bz) and immunomodulators (IMiDs) like lenalinomide (Len) are generally effective in newly diagnosed patients, some do not respond to this first-line therapy, and all others will eventually become drug resistant. We previously reported that inhibiting the Sec61 translocon with mycolactone synergizes with Bz to induce terminal unfolded protein response in MM cells, irrespective of their resistance to proteasome inhibition. Here, we examined how Sec61 blockade interferes with IMiD action and whether it overrides resistance to Len. With this aim, we knocked out the IMiD target CRBN in the MM1S cell line and a Bz-resistant subclone to generate Len- and Len/Bz-resistant daughters, respectively. Both the Len- and Len/Bz-resistant clones were susceptible to mycolactone toxicity, especially the doubly resistant one. Notably, the synergy between mycolactone and Bz was maintained in these two clones, and mycolactone also synergized with Len in the two Len-susceptible ones. Further, mycolactone enhanced the therapeutic efficacy of the Bz/Len combination in both mice engrafted with parental or double drug resistant MM1S. Together, these data consolidate the interest of Sec61 blockers as new anti-MM agents and reveal their potential for treatment of refractory or relapsed MM.

SAYSD1 senses UFMylated ribosome to safeguard co-translational protein translocation at the endoplasmic reticulum.

Translocon clogging at the endoplasmic reticulum (ER) as a result of translation stalling triggers ribosome UFMylation, activating translocation-associated quality control (TAQC) to degrade clogged substrates. How cells sense ribosome UFMylation to initiate TAQC is unclear. We conduct a genome-wide CRISPR-Cas9 screen to identify an uncharacterized membrane protein named SAYSD1 that facilitates TAQC. SAYSD1 associates with the Sec61 translocon and also recognizes both ribosome and UFM1 directly, engaging a stalled nascent chain to ensure its transport via the TRAPP complex to lysosomes for degradation. Like UFM1 deficiency, SAYSD1 depletion causes the accumulation of translocation-stalled proteins at the ER and triggers ER stress. Importantly, disrupting UFM1- and SAYSD1-dependent TAQC in Drosophila leads to intracellular accumulation of translocation-stalled collagens, defective collagen deposition, abnormal basement membranes, and reduced stress tolerance. Thus, SAYSD1 acts as a UFM1 sensor that collaborates with ribosome UFMylation at the site of clogged translocon, safeguarding ER homeostasis during animal development.

Mechanism of signal-anchor triage during early steps of membrane protein insertion.

Most membrane proteins use their first transmembrane domain, known as a signal anchor (SA), for co-translational targeting to the endoplasmic reticulum (ER) via the signal recognition particle (SRP). The SA then inserts into the membrane using either the Sec61 translocation channel or the ER membrane protein complex (EMC) insertase. How EMC and Sec61 collaborate to ensure SA insertion in the correct topology is not understood. Using site-specific crosslinking, we detect a pre-insertion SA intermediate adjacent to EMC. This intermediate forms after SA release from SRP but before ribosome transfer to Sec61. The polypeptide's N-terminal tail samples a cytosolic vestibule bordered by EMC3, from where it can translocate across the membrane concomitant with SA insertion. The ribosome then docks on Sec61, which has an opportunity to insert those SAs skipped by EMC. These results suggest that EMC acts between SRP and Sec61 to triage SAs for insertion during membrane protein biogenesis.