Endoplasmic reticulum stress and lipids in health and diseases.

The endoplasmic reticulum (ER) is a complex and dynamic organelle that regulates many cellular pathways, including protein synthesis, protein quality control, and lipid synthesis. When one or multiple ER roles are dysregulated and saturated, the ER enters a stress state, which, in turn, activates the highly conserved unfolded protein response (UPR). By sensing the accumulation of unfolded proteins or lipid bilayer stress (LBS) at the ER, the UPR triggers pathways to restore ER homeostasis and eventually induces apoptosis if the stress remains unresolved. In recent years, it has emerged that the UPR works intimately with other cellular pathways to maintain lipid homeostasis at the ER, and so does at cellular levels. Lipid distribution, along with lipid anabolism and catabolism, are tightly regulated, in part, by the ER. Dysfunctional and overwhelmed lipid-related pathways, independently or in combination with ER stress, can have reciprocal effects on other cellular functions, contributing to the development of diseases. In this review, we summarize the current understanding of the UPR in response to proteotoxic stress and LBS and the breadth of the functions mitigated by the UPR in different tissues and in the context of diseases.

A new combinatorial megaplasmid library assembly method designed to screen for minimal pathways by using SCRaMbLE.

Human proteins expressed in yeast are common to enhance protein production while the expression of functional human pathways remain challenging. Here, we propose a simple and economical high-throughput gene assembly method to create a yeast megaplasmid library from human cDNA to screen for minimal human functional pathways. We introduced artificial promoters followed by symmetric loxP sites into the megaplasmids using Golden Gate assembly coupled with streptavidin-bead-based purification. The isolated high molecular weight, randomly assembled cDNA megaplasmid library may be useful for high-throughput directed evolution experiments and may be adapted for use in other model organisms.

Vaccine for Diabetes-Where Do We Stand?

Diabetes is an endocrinological disorder with a rapidly increasing number of patients globally. Over the last few years, the alarming status of diabetes has become a pivotal factor pertaining to morbidity and mortality among the youth as well as middle-aged people. Current developments in our understanding related to autoimmune responses leading to diabetes have developed a cause for concern in the prospective usage of immunomodulatory agents to prevent diabetes. The mechanism of action of vaccines varies greatly, such as removing autoreactive T cells and inhibiting the interactions between immune cells. Currently, most developed diabetes vaccines have been tested in animal models, while only a few human trials have been completed with positive outcomes. In this review, we investigate the undergoing clinical trial studies for the development of a prototype diabetes vaccine.

Human PERK rescues unfolded protein response-deficient yeast cells.

Protein folding and quality control is tightly regulated at the endoplasmic reticulum (ER), and its disruption is associated with many diseases. In eukaryotes, the accumulation of unfolded protein in the ER is sensed by the three sensors, IRE1, PERK, and ATF6 to activate the unfolded protein response (UPR) to restore ER homeostasis. However, uncoupling the sensing of each sensor and their respective downstream pathways has been challenging as the absence of one is compensated by the remaining two sensors. Here, we report a fully functional human PERK (hPERK) chimeric protein expressed in Saccharomyces cerevisiae that could be used for high throughput screen to identify new PERK inhibitory or activating compounds as well as to characterize the PERK stress sensing mechanisms.

A high-throughput genetic screening protocol to measure lipid bilayer stress-induced unfolded protein response in Saccharomyces cerevisiae.

The endoplasmic reticulum (ER) stress is defined by the accumulation of unfolded proteins at the ER and perturbation at the ER membrane, known as lipid bilayer stress (LBS). In turn, ER stress triggers the unfolded protein response (UPR) to restore ER homeostasis. Here, we provide a modified protocol based on the synthetic genetic array analysis in Saccharomyces cerevisiae to identify genetic perturbations that induce the UPR by LBS. This method is adaptable to other canonical stress pathways. For complete details on the use and execution of this protocol, please refer to Ho et al. (2020), Jonikas et al. (2009) and Baryshnikova et al. (2010).

The yeast FIT2 homologs are necessary to maintain cellular proteostasis and membrane lipid homeostasis.

Lipid droplets (LDs) are implicated in conditions of lipid and protein dysregulation. The fat storage-inducing transmembrane (FIT; also known as FITM) family induces LD formation. Here, we establish a model system to study the role of the Saccharomyces cerevisiae FIT homologues (ScFIT), SCS3 and YFT2, in the proteostasis and stress response pathways. While LD biogenesis and basal endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) remain unaltered in ScFIT mutants, SCS3 was found to be essential for proper stress-induced UPR activation and for viability in the absence of the sole yeast UPR transducer IRE1 Owing to not having a functional UPR, cells with mutated SCS3 exhibited an accumulation of triacylglycerol within the ER along with aberrant LD morphology, suggesting that there is a UPR-dependent compensatory mechanism that acts to mitigate lack of SCS3 Additionally, SCS3 was necessary to maintain phospholipid homeostasis. Strikingly, global protein ubiquitylation and the turnover of both ER and cytoplasmic misfolded proteins is impaired in ScFITΔ cells, while a screen for interacting partners of Scs3 identifies components of the proteostatic machinery as putative targets. Together, our data support a model where ScFITs play an important role in lipid metabolism and proteostasis beyond their defined roles in LD biogenesis.This article has an associated First Person interview with the first author of the paper.

Stress sensor Ire1 deploys a divergent transcriptional program in response to lipid bilayer stress.

Membrane integrity at the endoplasmic reticulum (ER) is tightly regulated, and its disturbance is implicated in metabolic diseases. Using an engineered sensor that activates the unfolded protein response (UPR) exclusively when normal ER membrane lipid composition is compromised, we identified pathways beyond lipid metabolism that are necessary to maintain ER integrity in yeast and in C. elegans. To systematically validate yeast mutants that disrupt ER membrane homeostasis, we identified a lipid bilayer stress (LBS) sensor in the UPR transducer protein Ire1, located at the interface of the amphipathic and transmembrane helices. Furthermore, transcriptome and chromatin immunoprecipitation analyses pinpoint the UPR as a broad-spectrum compensatory response wherein LBS and proteotoxic stress deploy divergent transcriptional UPR programs. Together, these findings reveal the UPR program as the sum of two independent stress responses, an insight that could be exploited for future therapeutic intervention.