Adaptation to Endoplasmic Reticulum Stress in Candida albicans Relies on the Activity of the Hog1 Mitogen-Activated Protein Kinase

Adaptation to ER stress is linked to the pathogenicity of C. albicans. The fungus responds to ER stress primarily by activating the conserved Ire1-Hac1-dependent unfolded protein response (UPR) pathway. Subsequently, when ER homeostasis is re-established, the UPR is attenuated in a timely manner, a facet that is unexplored in C. albicans. Here, we show that C. albicans licenses the HOG (high-osmolarity glycerol) MAPK pathway for abating ER stress as evidenced by activation and translocation of Hog1 to the nucleus during tunicamycin-induced ER stress. We find that, once activated, Hog1 attenuates the activity of Ire1-dependent UPR, thus facilitating adaptation to ER stress. We use the previously established assay, where the disappearance of the UPR-induced spliced HAC1 mRNA correlates with the re-establishment of ER homeostasis, to investigate attenuation of the UPR in C. albicans. hog1Δ/Δ cells retain spliced HAC1 mRNA levels for longer duration reflecting the delay in attenuating Ire1-dependent UPR. Conversely, compromising the expression of Ire1 (ire1 DX mutant strain) results in diminished levels of phosphorylated Hog1, restating the cross-talk between Ire1 and HOG pathways. Phosphorylation signal to Hog1 MAP kinase is relayed through Ssk1 in response to ER stress as inactivation of Ssk1 abrogates Hog1 phosphorylation in C. albicans. Additionally, Hog1 depends on its cytosolic as well as nuclear activity for mediating ER stress-specific responses in the fungus. Our results show that HOG pathway serves as a point of cross-talk with the UPR pathway, thus extending the role of this signaling pathway in promoting adaptation to ER stress in C. albicans. Additionally, this study integrates this MAPK pathway into the little known frame of ER stress adaptation pathways in C. albicans.

SARS-CoV-2 ORF3a Induces Incomplete Autophagy via the Unfolded Protein Response

In the past year and a half, SARS-CoV-2 has caused 240 million confirmed cases and 5 million deaths worldwide. Autophagy is a conserved process that either promotes or inhibits viral infections. Although coronaviruses are known to utilize the transport of autophagy-dependent vesicles for the viral life cycle, the underlying autophagy-inducing mechanisms remain largely unexplored. Using several autophagy-deficient cell lines and autophagy inhibitors, we demonstrated that SARS-CoV-2 ORF3a was able to induce incomplete autophagy in a FIP200/Beclin-1-dependent manner. Moreover, ORF3a was involved in the induction of the UPR (unfolded protein response), while the IRE1 and ATF6 pathways, but not the PERK pathway, were responsible for mediating the ORF3a-induced autophagy. These results identify the role of the UPR pathway in the ORF3a-induced classical autophagy process, which may provide us with a better understanding of SARS-CoV-2 and suggest new therapeutic modalities in the treatment of COVID-19.

Non-canonical activation of the ER stress sensor ATF6 by Legionella pneumophila effectors

The intracellular bacterial pathogen Legionella pneumophila (L.p.) secretes ∼330 effector proteins into the host cell to sculpt an ER-derived replicative niche. We previously reported five L.p. effectors that inhibit IRE1, a key sensor of the homeostatic unfolded protein response (UPR) pathway. In this study, we discovered a subset of L.p. toxins that selectively activate the UPR sensor ATF6, resulting in its cleavage, nuclear translocation, and target gene transcription. In a deviation from the conventional model, this L.p.–dependent activation of ATF6 does not require its transport to the Golgi or its cleavage by the S1P/S2P proteases. We believe that our findings highlight the unique regulatory control that L.p. exerts upon the three UPR sensors and expand the repertoire of bacterial proteins that selectively perturb host homeostatic pathways.

The Role of the UPR Pathway in the Pathophysiology and Treatment of Bipolar Disorder

Bipolar disorder (BD) is a mood disorder that affects millions worldwide and is associated with severe mood swings between mania and depression. The mood stabilizers valproate (VPA) and lithium (Li) are among the main drugs that are used to treat BD patients. However, these drugs are not effective for all patients and cause serious side effects. Therefore, better drugs are needed to treat BD patients. The main barrier to developing new drugs is the lack of knowledge about the therapeutic mechanism of currently available drugs. Several hypotheses have been proposed for the mechanism of action of mood stabilizers. However, it is still not known how they act to alleviate both mania and depression. The pathology of BD is characterized by mitochondrial dysfunction, oxidative stress, and abnormalities in calcium signaling. A deficiency in the unfolded protein response (UPR) pathway may be a shared mechanism that leads to these cellular dysfunctions. This is supported by reported abnormalities in the UPR pathway in lymphoblasts from BD patients. Additionally, studies have demonstrated that mood stabilizers alter the expression of several UPR target genes in mouse and human neuronal cells. In this review, we outline a new perspective wherein mood stabilizers exert their therapeutic mechanism by activating the UPR. Furthermore, we discuss UPR abnormalities in BD patients and suggest future research directions to resolve discrepancies in the literature.

Screening The Efficacy of Melatonin On Neurodegeneration Mediated By ER Stress, Inflammation And Oxidative Damage

Neurodegeneration may be defined as a clinical condition wherein neurons gradually lose their structural integrity, viability, functional abilities and the damage inflicted upon the neurons is often irreversible. The number of elderly patients suffering from Neurodegenerative disorders is expected to rise tremendously over the next couple of years. Thus, there is an urgent need to delve into and study the underlying cause and mechanisms, so that we may be able to develop more effective therapeutic strategies and drugs and better understand the origin and progression of the disease.The various mechanisms that have been observed to contribute to neurodegeneration include aggregation and accumulation of misfolded proteins, impaired autophagy, oxidative damage, neuroinflammation, mitochondrial defects, increased SUMOylation of proteins, impaired UPR pathways, disruption of axonal transport.Melatonin, a neurohormone is involved in a variety of functions including scavenging free radicals, synchronizing the circadian rhythm, mitigating immune response.Melatonin has shown to modulate the UPR pathway ,antioxidant pathway through Nrf2 and inflammatory pathway through NFκB. The study aims to determine the efficacy of melatonin on neurodegeneration mediated by ER stress, inflammation and oxidative damage through in silico approaches. The molecular targets chosen were ATF6, XBP1, PERK, Nrf2, NFκB and they were docked against melatonin. Additionally various physiochemical analysis such as ADME were also carried out to determine its drug ability. The findings were that melatonin not only shows excellent interactions with the targets but also possess drug-like physicochemical properties that makes it a valuable choice for the treatment of neurodegenerative disorders.

Roles of High Osmolarity Glycerol and Cell Wall Integrity Pathways in Cadmium Toxicity in Saccharomyces cerevisiae

Cadmium is a carcinogen that can induce ER stress, DNA damage, oxidative stress and cell death. The yeast mitogen-activated protein kinase (MAPK) signalling pathways paly crucial roles in response to various stresses. Here, we demonstrate that the unfolded protein response (UPR) pathway, the high osmolarity glycerol (HOG) pathway and the cell wall integrity (CWI) pathway are all essential for yeast cells to defend against the cadmium-induced toxicity, including the elevated ROS and cell death levels induced by cadmium. We show that the UPR pathway is required for the cadmium-induced phosphorylation of HOG_MAPK Hog1 but not for CWI_MAPK Slt2, while Slt2 but not Hog1 is required for the activation of the UPR pathway through the transcription factors of Swi6 and Rlm1. Moreover, deletion of HAC1 and IRE1 could promote the nuclear accumulation of Hog1, and increase the cytosolic and bud neck localisation of Slt2, indicating crucial roles of Hog1 and Slt2 in regulating the cellular process in the absence of UPR pathway. Altogether, our findings highlight the significance of these two MAPK pathways of HOG and CWI and their interrelationship with the UPR pathway in responding to cadmium-induced toxicity in budding yeast.

Engineering of the unfolded protein response pathway in Pichia pastoris: enhancing production of secreted recombinant proteins

Folding and processing of proteins in the endoplasmic reticulum (ER) are major impediments in the production and secretion of proteins from Pichia pastoris (Komagataella sp.). Overexpression of recombinant genes can overwhelm the innate secretory machinery of the P. pastoris cell, and incorrectly folded proteins may accumulate inside the ER. To restore proper protein folding, the cell naturally triggers an unfolded protein response (UPR) pathway, which upregulates the expression of genes coding for chaperones and other folding-assisting proteins (e.g., Kar2p, Pdi1, Ero1p) via the transcription activator Hac1p. Unfolded/misfolded proteins that cannot be repaired are degraded via the ER-associated degradation (ERAD) pathway, which decreases productivity. Co-expression of selected UPR genes, along with the recombinant gene of interest, is a common approach to enhance the production of properly folded, secreted proteins. Such an approach, however, is not always successful and sometimes, protein productivity decreases because of an unbalanced UPR. This review summarizes successful chaperone co-expression strategies in P. pastoris that are specifically related to overproduction of foreign proteins and the UPR. In addition, it illustrates possible negative effects on the cell’s physiology and productivity resulting from genetic engineering of the UPR pathway. We have focused on Pichia’s potential for commercial production of valuable proteins and we aim to optimize molecular designs so that production strains can be tailored to suit a specific heterologous product. Key points • Chaperones co-expressed with recombinant genes affect productivity in P. pastoris. • Enhanced UPR may impair strain physiology and promote protein degradation. • Gene copy number of the target gene and the chaperone determine the secretion rate.

In vitro Anticancer Effects of JI017 on Two Prostate Cancer Cell Lines Involve Endoplasmic Reticulum Stress Mediated by Elevated Levels of Reactive Oxygen Species

Prostate cancer is the second most commonly diagnosed cancer, and prostate cancer is the second most common cause of cancer death in United States men after lung cancer. Many therapies are used to treat prostate cancer, and chemotherapy is one of the most relevant treatments. However, chemotherapy has many side effects, and repeated administration of chemotherapeutic agents leads to acquired resistance. Thus, new drugs with few side effects are needed. We investigated the molecular mechanism of action of JI017 in human prostate cancer cells. We identified an endoplasmic reticulum (ER) stress pathway that depended on the reactive oxygen species (ROS) pathway and played a crucial role in JI017-induced apoptosis. We measured cell viability by the MTS assay to determine the effect of JI017. Analysis of apoptosis, mitochondrial dysfunction, and cell cycle features was performed by flow cytometry. We used western blot and RT-PCR to measure the levels of the proteins of the unfolded protein response (UPR) pathway and apoptosis markers. Immunoprecipitation assay and transfection were used to determine the expression levels of proteins interacting with the pathways influenced by JI017 in prostate cancer cells. The anticancer effects induced by JI017 were evaluated. JI017 induced cell death that regulated apoptotic molecules and caused cell cycle arrest that inhibited the proliferation of cancer cells. Moreover, JI017 generated ROS. Accumulation of ROS caused ER stress through the PERK–eIF2α–CHOP and IRE1α-CHOP pathways. Furthermore, persistent activation of the UPR pathway induced by JI017 treatment triggered mitochondrial dysfunction, including dissipation of mitochondrial membrane potential, which activated intrinsic apoptotic pathway in human prostate cancer cells. The data indicated that N-acetyl-L-cysteine diminished apoptosis. We demonstrated that JI017 induced ER stress and cell death. Anticancer properties of JI017 in prostate cancer cells and in a human prostate cancer model involved ROS-mediated ER stress. Thus, JI017 treatment provides a new strategy for chemotherapy of prostate cancer.