The IRE1ß-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells.

Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1ß) with the luminal domain of IRE1ß deregulated basal IRE1 activity. The mucin-specific chaperone AGR2 repressed IRE1 activity in cells expressing the domain-swapped IRE1ß/α chimera, but had no effect on IRE1α. Introduction of the goblet cell-specific client MUC2 reversed AGR2-mediated repression of the IRE1ß/α chimera. In vitro, AGR2 actively de-stabilised the IRE1ß luminal domain dimer and formed a reversible complex with the inactive monomer. These features of the IRE1ß-AGR2 couple suggest that active repression of IRE1ß by a specialised mucin chaperone subordinates IRE1 activity to a proteostatic challenge unique to goblet cells, a challenge that is otherwise poorly recognised by the pervasive UPR transducers.

A comprehensive set of ER protein disulfide isomerase family members supports the biogenesis of proinflammatory interleukin 12 family cytokines.

Cytokines of the interleukin 12 (IL-12) family are assembled combinatorially from shared α and ß subunits. A common theme is that human IL-12 family α subunits remain incompletely structured in isolation until they pair with a designate ß subunit. Accordingly, chaperones need to support and control specific assembly processes. It remains incompletely understood, which chaperones are involved in IL-12 family biogenesis. Here, we site-specifically introduce photocrosslinking amino acids into the IL-12 and IL-23 α subunits (IL-12α and IL-23α) for stabilization of transient chaperone-client complexes for mass spectrometry. Our analysis reveals that a large set of endoplasmic reticulum chaperones interacts with IL-12α and IL-23α. Among these chaperones, we focus on protein disulfide isomerase (PDI) family members and reveal IL-12 family subunits to be clients of several incompletely characterized PDIs. We find that different PDIs show selectivity for different cysteines in IL-12α and IL-23α. Despite this, PDI binding generally stabilizes unassembled IL-12α and IL-23α against degradation. In contrast, α:ß assembly appears robust, and only multiple simultaneous PDI depletions reduce IL-12 secretion. Our comprehensive analysis of the IL-12/IL-23 chaperone machinery reveals a hitherto uncharacterized role for several PDIs in this process. This extends our understanding of how cells accomplish the task of specific protein assembly reactions for signaling processes. Furthermore, our findings show that cytokine secretion can be modulated by targeting specific endoplasmic reticulum chaperones.

Site-Specific Protein Labeling with Fluorophores as a Tool To Monitor Protein Turnover.

Proteins that terminally fail to acquire their native structure are detected and degraded by cellular quality control systems. Insights into cellular protein quality control are key to a better understanding of how cells establish and maintain the integrity of their proteome and of how failures in these processes cause human disease. Here we have used genetic code expansion and fast bio-orthogonal reactions to monitor protein turnover in mammalian cells through a fluorescence-based assay. We have used immune signaling molecules (interleukins) as model substrates and shown that our approach preserves normal cellular quality control, assembly processes, and protein functionality and works for different proteins and fluorophores. We have further extended our approach to a pulse-chase type of assay that can provide kinetic insights into cellular protein behavior. Taken together, this study establishes a minimally invasive method to investigate protein turnover in cells as a key determinant of cellular homeostasis.

COVID-19, MIS-C, and long COVID in pediatric patients: an update.

COVID-19, the disease caused by SARS-CoV-2, has been disruptive worldwide. It was primarily a respiratory disease that affected many of the medically vulnerable, but the true impact of postacute sequelae of SARS-CoV-2 (PASC), which has been demonstrated to involve all organ systems, is now coming to light. In addition, a new disease entity emerged, multisystem inflammatory syndrome in children (MIS-C), which has had significant morbidity and mortality associated with it. This issue reviews the presentation, evaluation, and management of patients with COVID-19, MIS-C, and PASC. Additionally, the current literature supporting public health measures, as well as COVID-19 vaccinations and their complications are discussed.

Spatial Proteomics of Single Hepatocytes with Multiplexed Data-Independent Acquisition (mDIA).

Spatially resolved mass spectrometry-based proteomics at single-cell resolution promises to provide insights into biological heterogeneity. We describe a protocol based on multiplexed data-independent acquisition (mDIA) with dimethyl labeling to enhance proteome depth, accuracy, and throughput while minimizing costs. It enables high-quality proteome analysis of single isolated hepatocytes and utilizes liver zonation for single-cell proteomics benchmarking. This adaptable, modular protocol will promote the use of single-cell proteomics in spatial biology.

A rapid, modular and marker-free chloroplast expression system for the green alga Chlamydomonas reinhardtii.

In search of alternative expression platforms heterologous protein production in microalgae has gained increasing importance in the last years. Particularly, the chloroplast of the green alga Chlamydomonas reinhardtii has been adopted to successfully express foreign proteins like vaccines and antibodies. However, when compared with other expression systems, the development of the algal chloroplast to a powerful production platform for recombinant proteins is still in its early stages. In an effort to further improve methods for a reliable and rapid generation of transplastomic Chlamydomonas strains we constructed the key plasmid pMM2 containing the psbA gene and a multiple cloning site for foreign gene insertion. The psbA gene allows a marker-free selection procedure using as a recipient the Fud7 strain of Chlamydomonas, which grows on media containing acetate as a carbon source, but is unable to grow photoautotrophically due to the lack of an intact psbA gene. Biolistic transformation of Fud7 with vectors containing this gene restores photoautotrophic growth and thus permits selection in the light on media without carbon sources and antibiotics. The multiple cloning site with a BsaI recognition sequence allows type IIs restriction enzyme-based modular cloning which rapidly generates new gene constructs without sequences, which could influence the expression and characteristics of the foreign protein. In order to demonstrate the feasibility of this approach, a codon optimized version of the gene for the bacterial protein MPT64 has been integrated into the plastome. Several strains with different promoter/UTR combinations show a stable expression of the HA tagged MPT64 protein in Chlamydomonas chloroplasts.

Effects of rare codon clusters on the expression of a high-turnover chloroplast protein in Chlamydomonas reinhardtii.

Expression of foreign proteins in chloroplasts has become an important field of plant genetic engineering. Optimized codon usage is generally thought to increase translational efficiency, but high speed translation of codon bias-adjusted mRNAs can also result in protein misfolding due to a lack of rare codons. In order to analyze the effect of rare codons on a native chloroplast protein in vivo, we modified the D1 subunit of photosystem II by fusing small peptides with different codons into a loop region which tolerates insertions without loss of function. Because of its high-turnover properties, the D1 protein represents an excellent test object to investigate the impact of rare codons on its translation. We choose codons for amino acids Arg, Leu, Ser, Ala and Gly which are rarely used and compared translation of the modified D1 proteins with the respective mutant proteins containing insertions with frequently used codons. Our data indicate that only rare Arg codons drastically affect synthesis of the D1 protein and cluster of rare Ser-codon can induce strategic ribosomal pausing sites.