Protein Synthesis In Endoplasmic Reticulum Research Articles

Multi-omics reveals the molecular mechanism of the combined toxic effects of PFOA and 4-HBP exposure in MCF-7 cells and the key player: mTORC1

With the discovery of evidence that many endocrine-disrupting chemicals (EDCs) in the environment influence human health, their toxic effects and mechanisms have become a hot topic of research. However, investigations into their endocrine-disrupting toxicity under combined binary exposure, especially the molecular mechanism of combined effects, have rarely been documented. In this study, two typical EDCs, perfluorooctanoic acid (PFOA) and 4-hydroxybenzophenone (4-HBP), were selected to examine their combined effects and molecular mechanism on MCF-7 cell proliferation at environmentally relevant exposure concentrations. We have successfully established a model to evaluate the binary combined toxic effects of endocrine disruptors, presenting combined effects in a simple and direct way. Results indicated that the combined effect changed from additive to synergistic from 1.25 × 10−8 M to 4 × 10−7 M. Metabolomics analyses suggested that exposure to PFOA and 4-HBP caused significant alterations in purine metabolism, arginine, and proline metabolism and had superimposed influences on metabolism. Enhanced combined effects were observed in glycine, serine, and threonine metabolic pathways compared to exposure to PFOS and 4-HBP alone. Additionally, the differentially expressed genes (DEGs) are primarily involved in Biological Processes, especially protein targeting the endoplasmic reticulum, and significantly impact the oxidative phosphorylation and thermogenesis-related KEGG pathway. By integrating metabolome and transcriptome analyses, PFOA and 4-HBP regulate purine metabolism, the TCA cycle, and endoplasmic reticulum protein synthesis in MCF-7 cells via mTORC1, which provides genetic material, protein, and energy for cell proliferation. Furthermore, molecular docking confirmed the ability of PFOA and 4-HBP to stably bind the estrogen receptor, indicating that they have different binding pockets. Collectively, these findings will offer new insights into understanding the mechanisms by which EDCs produce combined toxicity.

SMS2, a Novel Allele of OsINV3, Regulates Grain Size in Rice.

Grain size has an important effect on rice yield. Although several key genes that regulate seed size have been reported in rice, their molecular mechanisms remain unclear. In this study, a rice small grain size 2 (sms2) mutant was identified, and MutMap resequencing analysis results showed that a 2 bp insertion in the second exon of the LOC_Os02g01590 gene resulted in a grain length and width lower than those of the wild-type Teqing (TQ). We found that SMS2 encoded vacuolar acid invertase, a novel allele of OsINV3, which regulates grain size. GO and KEGG enrichment analyses showed that SMS2 was involved in endoplasmic reticulum protein synthesis, cysteine and methionine metabolism, and propionic acid metabolism, thereby regulating grain size. An analysis of sugar content in young panicles showed that SMS2 reduced sucrose, fructose, and starch contents, thus regulating grain size. A haplotype analysis showed that Hap2 of SMS2 had a longer grain and was widely present in indica rice varieties. Our results provide a new theoretical basis for the molecular and physiological mechanisms by which SMS2 regulates grain size.

The role of sphingolipids in endoplasmic reticulum stress.

The endoplasmic reticulum (ER) is an important intracellular compartment in eukaryotic cells and has diverse functions, including protein synthesis, protein folding, lipid metabolism and calcium homeostasis. ER functions are disrupted by various intracellular and extracellular stimuli that cause ER stress, including the inhibition of glycosylation, disulphide bond reduction, ER calcium store depletion, impaired protein transport to the Golgi, excessive ER protein synthesis, impairment of ER-associated protein degradation and mutated ER protein expression. Distinct ER stress signalling pathways, which are known as the unfolded protein response, are deployed to maintain ER homeostasis, and a failure to reverse ER stress triggers cell death. Sphingolipids are lipids that are structurally characterized by long-chain bases, including sphingosine or dihydrosphingosine (also known as sphinganine). Sphingolipids are bioactive molecules long known to regulate various cellular processes, including cell proliferation, migration, apoptosis and cell-cell interaction. Recent studies have uncovered that specific sphingolipids are involved in ER stress. This review summarizes the roles of sphingolipids in ER stress and human diseases in the context of pathogenic events.

Tailoring the Saccharomyces cerevisiae endoplasmic reticulum for functional assembly of terpene synthesis pathway

The endoplasmic reticulum (ER) is a dynamic organelle that synthesizes and folds proteins. An imbalance between the ER protein synthesis load and its folding capacity triggers the unfolded protein response, thereby restoring normal ER functions via size adjustment. Inspired by such inherent genetic programming events, we engineered Saccharomyces cerevisiae to expand the ER by overexpressing a key ER size regulatory factor, INO2. ER space expansion enhanced ER protein synthesis and folding capacity, and relieved metabolic constraints imposed by the limited enzyme abundance. Harnessing the yeast ER for metabolic engineering, we ultimately increased the production of squalene and cytochrome P450-mediated protopanaxadiol by 71-fold and 8-fold, compared to their respective control strains without overexpression of INO2. Furthermore, genome-wide transcriptome analysis of the ER-expanded strain revealed that the significant improvement in terpene production was associated with global rewiring of the metabolic network. Therefore, the yeast ER can be engineered as a specialized compartment for enhancing terpene production, representing new possibilities for the high-level production of other value-added chemicals.

Dengue Virus Selectively Annexes Endoplasmic Reticulum-Associated Translation Machinery as a Strategy for Co-opting Host Cell Protein Synthesis.

A primary question in dengue virus (DENV) biology is the molecular strategy for recruitment of host cell protein synthesis machinery. Here, we combined cell fractionation, ribosome profiling, and transcriptome sequencing (RNA-seq) to investigate the subcellular organization of viral genome translation and replication as well as host cell translation and its response to DENV infection. We report that throughout the viral life cycle, DENV plus- and minus-strand RNAs were highly partitioned to the endoplasmic reticulum (ER), identifying the ER as the primary site of DENV translation. DENV infection was accompanied by an ER compartment-specific remodeling of translation, where ER translation capacity was subverted from host transcripts to DENV plus-strand RNA, particularly at late stages of infection. Remarkably, translation levels and patterns in the cytosol compartment were only modestly affected throughout the experimental time course of infection. Comparisons of ribosome footprinting densities of the DENV plus-strand RNA and host mRNAs indicated that DENV plus-strand RNA was only sparsely loaded with ribosomes. Combined, these observations suggest a mechanism where ER-localized translation and translational control mechanisms, likely cis encoded, are used to repurpose the ER for DENV virion production. Consistent with this view, we found ER-linked cellular stress response pathways commonly associated with viral infection, namely, the interferon response and unfolded protein response, to be only modestly activated during DENV infection. These data support a model where DENV reprograms the ER protein synthesis and processing environment to promote viral survival and replication while minimizing the activation of antiviral and proteostatic stress response pathways.IMPORTANCE DENV, a prominent human health threat with no broadly effective or specific treatment, depends on host cell translation machinery for viral replication, immune evasion, and virion biogenesis. The molecular mechanism by which DENV commandeers the host cell protein synthesis machinery and the subcellular organization of DENV replication and viral protein synthesis is poorly understood. Here, we report that DENV has an almost exclusively ER-localized life cycle, with viral replication and translation largely restricted to the ER. Surprisingly, DENV infection largely affects only ER-associated translation, with relatively modest effects on host cell translation in the cytosol. DENV RNA translation is very inefficient, likely representing a strategy to minimize disruption of ER proteostasis. Overall these findings demonstrate that DENV has evolved an ER-compartmentalized life cycle; thus, targeting the molecular signatures and regulation of the DENV-ER interaction landscape may reveal strategies for therapeutic intervention.

A novel heterozygous mutation of the WFS1 gene leading to constitutive endoplasmic reticulum stress is the cause of Wolfram syndrome

Wolfram syndrome (WS) is a disorder characterized by the association of insulin-dependent diabetes mellitus (DM), diabetes insipidus, deafness, and optic nerve atrophy. WS is caused by WFS1 mutations encoding WFS1 protein expressed in endoplasmic reticulum (ER). During ER protein synthesis, misfolded and unfolded proteins accumulate, known as "ER stress". This is attenuated by the unfolded protein response (UPR), which recovers and maintains ER functions. Because WFS1 is a UPR component, mutant WFS1 might cause unresolvable ER stress conditions and cell apoptosis, the major causes underlying WS symptoms. We encountered an 11-month-old Japanese female WS patient with insulin-dependent DM, congenital cataract and severe bilateral hearing loss. Analyze the WFS1 and functional consequence of the patient WFS1 in vitro. The patient WFS1 contained a heterozygous 4 amino acid in-frame deletion (p.N325_I328del). Her mutant WFS1 increased GRP78 and ATF6α promoter activities in the absence of thapsigargin, indicating constitutive ER stress and nuclear factor of activated T-cell reporter activity, reflecting elevated cytosolic Ca2+ signals. Mutant transfection into cells reduced mRNA expression levels of sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase 2b (SERCA2b) compared with wild type. Because SERCA2b is required for ER and cytoplasmic Ca2+ homeostasis, decreased SERCA2b expression might affect ER Ca2+ efflux, causing cell apoptosis. A novel heterozygous mutation of WFS1 induced constitutive ER stress through ATF6α activation and ER Ca2+ efflux, resulting in cell apoptosis. These results provide new insights into the roles of WFS1 in UPR and mechanism of monogenic DM.

Divergent Regulation of Protein Synthesis in the Cytosol and Endoplasmic Reticulum Compartments of Mammalian Cells

In eukaryotic cells, mRNAs encoding signal sequence-bearing proteins undergo translation-dependent trafficking to the endoplasmic reticulum (ER), thereby restricting secretory and integral membrane protein synthesis to the ER compartment. However, recent studies demonstrating that mRNAs encoding cytosolic/nucleoplasmic proteins are represented on ER-bound polyribosomes suggest a global role for the ER in cellular protein synthesis. Here, we examined the steady-state protein synthesis rates and compartmental distribution of newly synthesized proteins in the cytosol and ER compartments. We report that ER protein synthesis rates exceed cytosolic protein synthesis rates by 2.5- to 4-fold; yet, completed proteins accumulate to similar levels in the two compartments. These data suggest that a significant fraction of cytosolic proteins undergo synthesis on ER-bound ribosomes. The compartmental differences in steady-state protein synthesis rates correlated with a divergent regulation of the tRNA aminoacylation/deacylation cycle. In the cytosol, two pathways were observed to compete for aminoacyl-tRNAs-protein synthesis and aminoacyl-tRNA hydrolysis-whereas on the ER tRNA deacylation is tightly coupled to protein synthesis. These findings identify a role for the ER in global protein synthesis, and they suggest models where compartmentalization of the tRNA acylation/deacylation cycle contributes to the regulation of global protein synthesis rates.