Human selenoprotein S (selenos) is part of the integrated cellular stress response and linked to protein quality control and signaling pathways. Consequently, genetic polymorphisms of selenos are associated with increased risk for diabetes, dyslipidemia, and cardiovascular diseases. Determining the specific roles of selenos in these cellular pathways and diseases has been challenging, as selenos associates with a wide range of protein complexes. Thus, to map the cellular functions of selenos and uncover their interconnections, we used affinity purification and in vivo crosslinking to stabilize transient protein interactions, followed by proteomics to record the resulting selenos interactome. Through mapping of selenos protein partners, we found evidence that selenos associates with complexes responsible for the insertion of membrane proteins into the ER bilayer and their connected quality control components. Furthermore, selenos is also part of metabolic, trafficking, and mitochondrial pathways. Notably, proteins involved in translation preferentially associate with selenos when its C-terminal intrinsically disordered segment containing the redox-active motif is accessible. Together, these results identify the C-terminal redox loop of selenos as a central interaction hub connecting translation with ER membrane protein biogenesis and quality control.

Human selenoprotein S (selenos) is part of the integrated cellular stress response and linked to protein quality control and signaling pathways. Consequently, genetic polymorphisms of selenos are associated with increased risks for diabetes, dyslipidemia, and cardiovascular diseases. Determining the specific roles of selenos in these cellular pathways and diseases has been challenging, as selenos associates with a wide range of protein complexes. Thus, to map the cellular functions of selenos and uncover their interconnections, we used affinity purification and in vivo crosslinking to stabilize transient protein interactions, followed by proteomics to record the resulting selenos interactome. Through mapping of selenos protein partners, we found evidence that selenos associates with complexes responsible for the insertion of membrane proteins into the endoplasmic reticulum (ER) bilayer and their connected quality control components. Furthermore, selenos is also part of metabolic, trafficking, and mitochondrial pathways. Notably, proteins involved in translation preferentially associate with selenos when its C-terminal intrinsically disordered segment containing the redox-active motif is accessible. Together, these results identify the C-terminal redox loop of selenos as a central interaction hub connecting translation with ER membrane protein biogenesis and quality control.

Selenoprotein K (selenok) is a small, disordered membrane protein associated with the endoplasmic reticulum (ER) that is involved in protein palmitoylation and protein quality control. Through these processes, it influences calcium homeostasis, cellular migration, and phagocytosis. Thus, it is implicated in cancer, neurodegenerative diseases, and autophagy. So far, selenok has been considered a single-pass membrane protein whose N-terminus is in the ER lumen while its C-terminus, which contains the reactive selenocysteine, is in the cytoplasm. Here, we show that selenok is, in fact, a peripheral membrane protein that is anchored to the cytoplasmic side of the ER membrane. We demonstrate, using immunofluorescence microscopy and the substituted cysteine accessibility method in combination with selective membrane permeabilization, that both selenok's N- and C-terminus are in the cytoplasm. Using the same techniques, we demonstrate that, in contrast, selenoprotein S (selenos), a functionally related member of the selenoprotein family, is a transmembrane protein with a cytoplasmic C-terminus and an N-terminus exposed to the ER lumen. The findings that selenok is a peripheral membrane protein and that its N- and C-terminal segments, along with the hydrophilic side of its amphipathic α-helix, are exposed to the cytoplasm, imply that they can interact with cytoplasmic extramembranous regions of ER-residing membrane proteins and soluble protein partners. Selenok is predicted to possess multiple SLiMs (short linear motifs) involved in protein interactions, and its peripheral topology suggests that all these motifs, including those located within the amphipathic α-helix, are exposed and accessible to cytoplasmic-accessible partners.

CryoEM democratization is hampered by access to costly plunge-freezing supplies. We introduce methods, called CryoCycle, for reliably blotting, vitrifying, and reusing clipped cryoEM grids. We demonstrate that vitreous ice may be produced by plunging clipped grids with purified proteins into liquid ethane and that clipped grids may be reused several times for different protein samples. Furthermore, we demonstrate the vitrification of thin areas of cells prepared on gold-coated, pre-clipped grids.

Selenoprotein K (selenok) is linked to the integrated stress response, which helps cells combat stressors and regain normal function. The selenoprotein contains numerous protein interaction hubs and post-translational modification sites and is involved in protein palmitoylation, vesicle trafficking, and the resolution of ER stress. Anchored to the endoplasmic reticulum (ER) membrane, selenok interacts with protein partners to influence their stability, localization, and trafficking, impacting various cellular functions such as calcium homeostasis, cellular migration, phagocytosis, gene expression, and immune response. Consequently, selenok expression level is linked to cancer and neurodegenerative diseases.Because it contains the reactive amino acid selenocysteine, selenok is likely to function as an enzyme. However, highly unusual for enzymes, the protein segment containing the selenocysteine lacks a stable secondary or tertiary structure, yet it includes multiple interaction sites for protein partners and post-translational modifications. Currently, the reason(s) for the presence of the rare selenocysteine in selenok is not known. Furthermore, of selenok’s numerous interaction sites, only some have been sufficiently characterized, leaving many of selenok’s potential protein partners to be discovered. In this review, we explore selenok's role in various cellular pathways and its impact on human health, thereby highlighting the links between its diverse cellular functions.

Selenoprotein S (selenos), a small transmembrane protein, is linked to an increased risk of diseases such as cancer, cardiovascular issues, diabetes, and thyroid disorders. To deepen our understanding of selenos' functions, we are focusing on its role in mitigating endoplasmic reticulum (ER) stress. ER stress results in the accumulation of misfolded proteins within the ER membrane and lumen. Selenos actively participates in endoplasmic reticulum-associated degradation (ERAD), a process responsible for identifying misfolded proteins, extracting them from the ER, and degrading them in the cytosol.

NMR spectroscopy has been applied to virtually all sites within proteins and biomolecules; however, the observation of sulfur sites remains very challenging. Recent studies have examined 77Se as a replacement for sulfur and applied 77Se NMR in both the solution and solid states. As a spin-1/2 nuclide, 77Se is attractive as a probe of sulfur sites, and it has a very large chemical shift range (due to a large chemical shift anisotropy), which makes it potentially very sensitive to structural and/or binding interactions as well as dynamics. Despite being a spin-1/2 nuclide, there have been rather limited studies of 77Se, and the ability to use 1H-indirect detection has been sparse. Some examples exist, but in the absence of a directly bonded, nonexchangeable 1H, these have been largely limited to smaller molecules. We develop and illustrate approaches using double-labeling of 13C and 77Se in proteins that enable more sensitive triple-resonance schemes via multistep coherence transfers and 1H-detection. These methods require specialized hardware and decoupling schemes, which we developed and will be discussed.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) replicates and evades detection using ER membranes and their associated protein machinery. Among these hijacked human proteins is selenoprotein S (selenos). This selenoprotein takes part in the protein quality control, signaling, and the regulation of cytokine secretion. While the role of selenos in the viral life cycle is not yet known, it has been reported to interact with SARS-CoV-2 nonstructural protein 7 (nsp7), a viral protein essential for the replication of the virus. We set to study whether selenos and nsp7 interact directly and if they can still bind when nsp7 is bound to the replication and transcription complex of the virus. Using biochemical assays, we show that selenos binds directly to nsp7. In addition, we found that selenos can bind to nsp7 when it is in a complex with the coronavirus's minimal replication and transcription complex, comprised of nsp7, nsp8, and the RNA-dependent RNA polymerase nsp12. In addition, through crosslinking experiments, we mapped the interaction sites of selenos and nsp7 in the replication complex and showed that the hydrophobic segment of selenos is essential for binding to nsp7. This arrangement leaves an extended helix and the intrinsically disordered segment of selenos-including the reactive selenocysteine-exposed and free to potentially recruit additional proteins to the replication and transcription complex.

Sulfur-containing sites in proteins are of great importance for both protein structure and function, including enzymatic catalysis, signaling pathways, and recognition of ligands and protein partners. Selenium-77 is an NMR active spin-1/2 nucleus that shares many physiochemical properties with sulfur and can be readily introduced into proteins at sulfur sites without significant perturbations to the protein structure. The sulfur-containing amino acid methionine is commonly found at protein–protein or protein–ligand binding sites. Its selenium-containing counterpart, selenomethionine, has a broad chemical shift dispersion useful for NMR-based studies of complex systems. Methods such as (1H)-77Se-13C double cross polarization or {77Se}-13C REDOR could be valuable to map the local environment around selenium sites in proteins but have not been demonstrated to date. In this work, we explore these dipolar transfer mechanisms for structural characterization of the GB1 V39SeM variant of the model protein GB1 and demonstrate that 77Se-13C based correlations can be used to map the local environment around selenium sites in proteins. We have found that the general detection limit is ~ 5 Å, but longer range distances up to ~ 7 Å can be observed as well. This study establishes a framework for the future characterization of selenium sites at protein–protein or protein–ligand binding interfaces.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) uses endoplasmic reticulum membranes and associated human proteins for its replication and to evade detection. One human protein recruited by several viral proteins is selenoprotein S, which takes part in the endoplasmic reticulum protein degradation pathway, NFkB signaling, and cytokines secretion. A key interaction was reported with SARS-CoV-2 non-structural protein (nsp7), which is essential for virus replication. However, it was unclear whether selenoprotein S and nsp7 interact directly and whether the interaction is possible when nsp7 forms a complex with the other components of the virus's replication machinery. We used biochemical assays to show that selenoprotein S binds nsp7, including when nsp7 is in complex with the coronavirus's RNA-dependent RNA polymerase. This places selenoprotein S at the heart of the coronavirus's replication complex and marks it as the first human protein shown to directly interact with the viral replication complex. Cross-linking experiments were employed to map the interactions of selenoprotein S and nsp7 in the replication complex. We show that the hydrophobic segment of selenoprotein S is essential for binding nsp7. This arrangement leaves an extended helix and the intrinsically disordered region of selenoprotein S exposed and free to recruit additional proteins to the complex.

Selenoprotein S (selenos) is a small, intrinsically disordered membrane protein that is associated with various cellular functions, such as inflammatory processes, cellular stress response, protein quality control, and signaling pathways. It is primarily known for its contribution to the ER-associated degradation (ERAD) pathway, which governs the extraction of misfolded proteins or misassembled protein complexes from the ER to the cytosol for degradation by the proteasome. However, selenos's other cellular roles in signaling are equally vital, including the control of transcription factors and cytokine levels. Consequently, genetic polymorphisms of selenos are associated with increased risk for diabetes, dyslipidemia, and cardiovascular diseases, while high expression levels correlate with poor prognosis in several cancers. Its inhibitory role in cytokine secretion is also exploited by viruses. Since selenos binds multiple protein complexes, however, its specific contributions to various cellular pathways and diseases have been difficult to establish. Thus, the precise cellular functions of selenos and their interconnectivity have only recently begun to emerge. This review aims to summarize recent insights into the structure, interactome, and cellular roles of selenos.

Selenoproteins are a family of enzymes that employ the rare amino acid selenocysteine to catalyze chemical reactions. Among them, selenoprotein S stands out because the selenocysteine is positioned in an intrinsically disordered segment. The physiological function of this enzyme is unknown, although it was shown to take part in ER homeostasis by mediating protein degradation and may also have a role in vesicle trafficking, lipid metabolism, and management of oxidative stress. To elucidate its function in vesicle trafficking, we demonstrate that selenoprotein S binds the nucleotide exchange regulator of small GTPases, SmgGDS, a regulator of the Ras and Rho family members. Curiously, both selenoprotein S and SmgGDS are hijacked by non-structural proteins of SARS-CoV-2, along with other proteins involved in maintaining ER homeostasis and regulation of the secretory pathway. To investigate this link, we have characterized the interactions between selenoprotein S and SmgGDS. We show that the interaction requires the hydrophobic segment of selenoprotein S, previously thought to be transmembrane. We describe biochemical assays to examine the putative role of selenoprotein S in modulating SmgGDS function and whether it accelerates the rate release of GTPases from SmgGDS.

The evolutionarily conserved leucine rich repeat (LRR) protein domain is a unique structural motif found in many viral, bacterial, archaeal, and eukaryotic proteins. The LRR domain serves many roles, including being a signaling domain and a pathogen recognition receptor. In the human innate immune system, it serves an essential role by recognizing fragments of bacterial cell walls. Interestingly, the human fungal pathogen Candida albicans also uses an LRR domain-containing protein, Cyrp1, to sense bacterial cell wall fragments. However, the dynamics of signaling and detection of bacterial peptidoglycan fragments by the LRR of Cyr1p remains poorly characterized. Here we develop optimal recombinant expression workflows and provide characterization of the entire region of the LRR domain of Cyr1p as a peripheral membrane protein. Using a newly designed peptidoglycan enrichment bead assay, we demonstrate that this domain can bind bacterial peptidoglycan fragments under native conditions. The new membrane-associated Cyr1p-LRR construct sets the stage for the development of antifungal agents via high-throughput campaigns to inhibit cell wall-Cyr1p interactions.

Through known association with other proteins, human selenoprotein K (selenok) is currently implicated in the palmitoylation of proteins, degradation of misfolded proteins, innate immune response, and the life cycle of SARS-CoV-2 virus. However, neither the catalytic function of selenok’s selenocysteine (Sec), which, curiously, resides in an intrinsically disordered protein segment nor selenok’s specific role in these pathways are known to date. This report casts these questions in a new light as it describes that selenok is able -both in vitro and in vivo- to cleave some of its own peptide bonds. The cleavages not only release selenok segments that contain its reactive Sec, but as the specific cleavage sites were identified, they proved to cluster tightly near sites through which selenok interacts with protein partners. Furthermore, it is shown that selenok’s cleavage activity is neither restricted to itself nor promiscuous but selectively extends to at least one of its protein partners. Together, selenok’s cleavage ability and its features have all hallmarks of a regulatory mechanism that could play a central role in selenok’s associations with other proteins and its cellular functions overall.