Novel Pathogenic de novo INS p.T97P Variant Presenting with Severe Neonatal DKA

Pathogenic INS gene mutations are causative for Mutant INS-gene-induced Diabetes of Youth (MIDY). We characterize a novel de novo heterozygous INS gene mutation (c.289A>C, p.T97P) that presented in an autoantibody-negative 5-month-old male infant with severe diabetic ketoacidosis. In silico pathogenicity prediction tools provided contradictory interpretations, while structural modeling indicated a deleterious effect on proinsulin folding. Transfection of wildtype and INS p.T97P expression and luciferase reporter constructs demonstrated elevated intracellular mutant proinsulin levels and dramatically impaired proinsulin/insulin and luciferase secretion. Notably, proteasome inhibition partially and selectively rescued INS p.T97P-derived luciferase secretion. Additionally, expression of INS p.T97P caused increased intracellular proinsulin aggregate formation and XBP-1s protein levels, consistent with induction of endoplasmic reticulum stress. We conclude that INS p.T97P is a newly identified pathogenic A-chain variant that is causative for MIDY via disruption of proinsulin folding and processing with induction of the endoplasmic reticulum stress response.

Characterization and modulation of endoplasmic reticulum stress response target genes in Kluyveromyces marxianus to improve secretory expressions of heterologous proteins

Background Kluyveromyces marxianus is a promising cell factory for producing bioethanol and that raised a demand for a high yield of heterologous proteins in this species. Expressions of heterologous proteins usually lead to the accumulation of misfolded or unfolded proteins in the lumen of the endoplasmic reticulum (ER) and then cause ER stress. To cope with this problem, a group of ER stress response target genes (ESRTs) are induced, mainly through a signaling network called unfolded protein response (UPR). Characterization and modulation of ESRTs direct the optimization of heterologous expressions. However, ESRTs in K. marxianus have not been identified so far. Results In this study, we characterized the ER stress response in K. marxianus for the first time, by using two ER stress-inducing reagents, dithiothreitol (DTT) and tunicamycin (TM). Results showed that the Kar2–Ire1–Hac1 pathway of UPR is well conserved in K. marxianus. About 15% and 6% of genes were upregulated during treatment of DTT and TM, respectively. A total of 115 upregulated genes were characterized as ESRTs, among which 97 genes were identified as UPR target genes and 37 UPR target genes contained UPR elements in their promoters. Genes related to carbohydrate metabolic process and actin filament organization were identified as new types of UPR target genes. A total of 102 ESRTs were overexpressed separately in plasmids and their effects on productions of two different lignocellulolytic enzymes were systematically evaluated. Overexpressing genes involved in carbohydrate metabolism, including PDC1, PGK and VID28, overexpressing a chaperone gene CAJ1 or overexpressing a reductase gene MET13 substantially improved secretion expressions of heterologous proteins. Meanwhile, overexpressing a novel gene, KLMA_50479 (named ESR1), as well as overexpressing genes involved in ER-associated protein degradation (ERAD), including HRD3, USA1 andYET3, reduced the secretory expressions. ESR1 and the aforementioned ERAD genes were deleted from the genome. Resultant mutants, except the yet3Δ mutant, substantially improved secretions of three different heterologous proteins. During the fed-batch fermentation, extracellular activities of an endoxylanase and a glucanase in hrd3Δ cells improved by 43% and 28%, respectively, compared to those in wild-type cells. Conclusions Our results unveil the transcriptional scope of the ER stress response in K. marxianus and suggest efficient ways to improve productions of heterologous proteins by manipulating expressions of ESRTs.

A primary effect of palmitic acid on mouse oocytes is the disruption of the structure of the endoplasmic reticulum.

Exposure of mouse oocytes to saturated fatty acids such as palmitic acid has been shown to increases lipid content and cause an endoplasmic reticulum stress response and changes in the mitochondrial redox state. The links between these changes, or whether they are prevented by mono-unsaturated fatty acids such as oleic acid is unclear. Here, we have investigated the effects of fatty acids on mouse oocytes, that are maturated in vitro, using coherent anti-Stokes Raman scattering and two-photon fluorescence microscopy. When oocytes were matured in the presence of palmitic acid there were changes in the aggregation pattern and size of lipid droplets. Maturation in palmitic acid alone also caused a distinctive disruption of the endoplasmic reticulum structure. This effect was prevented by incubation of oleic with palmitic acid. In contrast, maturation of mouse oocytes in medium containing palmitic acid was not associated with any significant change in the redox state of mitochondria or the Ca2+ content of intracellular stores. These data suggest that a primary effect of saturated fatty acids such as PA on oocytes, is to disrupt the structure of the endoplasmic reticulum and this is not due to any other effect on mitochondria or Ca2+ stores.

Comprehensive Analysis of the Transcriptome-Wide m6A Methylation Modification Difference in Liver Fibrosis Mice by High-Throughput m6A Sequencing

N6-Methyladenosine (m6A), a unique and common mRNA modification method in eukaryotes, is involved in the occurrence and development of many diseases. Liver fibrosis (LF) is a common response to chronic liver injury and may lead to cirrhosis and even liver cancer. However, the involvement of m6A methylation in the development of LF is still unknown. In this study, we performed a systematic evaluation of hepatic genome-wide m6A modification and mRNA expression by m6A-seq and RNA-seq using LF mice. There were 3,315 genes with significant differential m6A levels, of which 2,498 were hypermethylated and 817 hypomethylated. GO and KEGG analyses illustrated that differentially expressed m6A genes were closely correlated with processes such as the endoplasmic reticulum stress response, PPAR signaling pathway and TGF-β signaling pathway. Moreover, a total of 90 genes had both a significant change in the m6A level and mRNA expression shown by joint analysis of m6A-seq and RNA-seq. Hence, the critical elements of m6A modification, including methyltransferase WTAP, demethylases ALKBH5 and binding proteins YTHDF1 were confirmed by RT-qPCR and Western blot. In an additional cell experiment, we also observed that the decreased expression of WTAP induced the development of LF as a result of promoting hepatic stellate cell (HSC) activation. Therefore, this study revealed unique differential m6A methylation patterns in LF mice and suggested that m6A methylation was associated with the occurrence and course of LF to some extent.

A Melanin-like Nanoenzyme for Acute Lung Injury Therapy via Suppressing Oxidative and Endoplasmic Reticulum Stress Response

Nanoenzyme-mediated catalytic activity is emerging as a novel strategy for reactive oxygen species (ROS) scavenging in acute lung injury (ALI) treatment. However, one of the main hurdles for these metal-containing nanoenzymes is their potential toxicity and single therapeutic mechanism. Herein, we uncovered a melanin-like nanoparticles derived from the self-polymerization of 1,8-dihydroxynaphthalene (PDH nanoparticles), showing a significant anti-inflammation therapeutic effect on ALI mice. The prepared PDH nanoparticles rich in phenol groups could not only act as radical scavengers to alleviate oxidative stress but could also chelate calcium overload to suppress the endoplasmic reticulum stress response. As revealed by the therapeutic effect in vivo, PDH nanoparticles significantly prohibited neutrophil infiltration and the secretion of proinflammatory cytokines (TNF-α and IL-6), thus improving the inflammatory cascade in the ALI model. Above all, our work provides an effective anti-inflammatory nanoplatform by using the inherent capability of melanin-like nanoenzymes, proposing the potential application prospects of these melanin-like nanoparticles for acute inflammation-induced injury treatment.

MANF: A Novel Endoplasmic Reticulum Stress Response Protein—The Role in Neurological and Metabolic Disorders

The mesencephalic astrocyte-derived neurotrophic factor (MANF), also named as arginine-rich protein (ARP) or arginine-rich mutated in early-stage tumors (ARMET), is a novel evolutionary conserved protein related to unfolded protein response. Growing evidence suggests that MANF critically involves in many ER stress-related diseases with a protective effect. Here, we review the function of MANF based on its structure in neurological and metabolic disorders and summarize its potential applications in disease diagnosis and therapies.

XBP1 regulates the protumoral function of tumor-associated macrophages in human colorectal cancer

Macrophages are among the most abundant immune cells in colorectal cancer (CRC). Re-educating tumor-associated macrophages (TAMs) to switch from protumoral to anti-tumoral activity is an attractive treatment strategy that warrants further investigation. However, little is known about the key pathway that is activated in TAMs. In this study, infitrating CD206+ TAMs in CRC were sorted and subjected to RNA-seq analysis. Differentially expressed genes were found to be enriched in unfolded protein response/endoplasmic reticulum stress response processes, and XBP1 splicing/activation was specifically observed in TAMs. XBP1 activation in TAMs promoted the growth and metastasis of CRC. Ablation of XBP1 inhibited the expression of the pro-tumor cytokine signature of TAMs, including IL-6, VEGFA, and IL-4. Simultaneously, XBP1 depletion could directly inhibit the expression of SIRPα and THBS1, thereby blocking “don’t eat me” recognition signals and enhancing phagocytosis. Therapeutic XBP1 gene editing using AAV2-sgXBP1 enhanced the anti-tumor activity. Together, XBP1 activation in TAMs drives CRC progression by elevating pro-tumor cytokine expression and secretion, as well as inhibiting macrophage phagocytosis. Targeting XBP1 signaling in TAMs may be a potential strategy for CRC therapy.