Insights and Perspectives on the Role of Proteostasis and Heat Shock Proteins in Fungal Infections.

Fungi are a diverse group of eukaryotic organisms that infect humans, animals, and plants. To successfully colonize their hosts, pathogenic fungi must continuously adapt to the host's unique environment, e.g., changes in temperature, pH, and nutrient availability. Appropriate protein folding, assembly, and degradation are essential for maintaining cellular homeostasis and survival under stressful conditions. Therefore, the regulation of proteostasis is crucial for fungal pathogenesis. The heat shock response (HSR) is one of the most important cellular mechanisms for maintaining proteostasis. It is activated by various stresses and regulates the activity of heat shock proteins (HSPs). As molecular chaperones, HSPs participate in the proteostatic network to control cellular protein levels by affecting their conformation, location, and degradation. In recent years, a growing body of evidence has highlighted the crucial yet understudied role of stress response circuits in fungal infections. This review explores the role of protein homeostasis and HSPs in fungal pathogenicity, including their contributions to virulence and host-pathogen interactions, as well as the concerted effects between HSPs and the main proteostasis circuits in the cell. Furthermore, we discuss perspectives in the field and the potential for targeting the components of these circuits to develop novel antifungal therapies.

Negative Modulation of Macroautophagy by Stabilized HERPUD1 is Counteracted by an Increased ER-Lysosomal Network With Impact in Drug-Induced Stress Cell Survival.

Macroautophagy and the ubiquitin proteasome system work as an interconnected network in the maintenance of cellular homeostasis. Indeed, efficient activation of macroautophagy upon nutritional deprivation is sustained by degradation of preexisting proteins by the proteasome. However, the specific substrates that are degraded by the proteasome in order to activate macroautophagy are currently unknown. By quantitative proteomic analysis we identified several proteins downregulated in response to starvation independently of ATG5 expression. Among them, the most significant was HERPUD1, an ER membrane protein with low expression and known to be degraded by the proteasome under normal conditions. Contrary, under ER stress, levels of HERPUD1 increased rapidly due to a blockage in its proteasomal degradation. Thus, we explored whether HERPUD1 stability could work as a negative regulator of autophagy. In this work, we expressed a version of HERPUD1 with its ubiquitin-like domain (UBL) deleted, which is known to be crucial for its proteasome degradation. In comparison to HERPUD1-WT, we found the UBL-deleted version caused a negative role on basal and induced macroautophagy. Unexpectedly, we found stabilized HERPUD1 promotes ER remodeling independent of unfolded protein response activation observing an increase in stacked-tubular structures resembling previously described tubular ER rearrangements. Importantly, a phosphomimetic S59D mutation within the UBL mimics the phenotype observed with the UBL-deleted version including an increase in HERPUD1 stability and ER remodeling together with a negative role on autophagy. Moreover, we found UBL-deleted version and HERPUD1-S59D trigger an increase in cellular size, whereas HERPUD1-S59D also causes an increased in nuclear size. Interestingly, ER remodeling by the deletion of the UBL and the phosphomimetic S59D version led to an increase in the number and function of lysosomes. In addition, the UBL-deleted version and phosphomimetic S59D version established a tight ER-lysosomal network with the presence of extended patches of ER-lysosomal membrane-contact sites condition that reveals an increase of cell survival under stress conditions. Altogether, we propose stabilized HERPUD1 downregulates macroautophagy favoring instead a closed interplay between the ER and lysosomes with consequences in drug-cell stress survival.

The Endoplasmic Reticulum Role in the Plant Response to Abiotic Stress.

The endoplasmic reticulum (ER) is the organelle where one third of the proteins of a cell are synthetized. Several of these proteins participate in the signaling and response of cells, tissues, or from the organism to the environment. To secure the proper synthesis and folding of these proteins, or the disposal of unfolded or misfolded proteins, the ER has different mechanisms that interact and regulate each other. These mechanisms are known as the ER quality control (ERQC), ER-associated degradation (ERAD) and the unfolded protein response (UPR), all three participants of the maintenance of ER protein homeostasis or proteostasis. Given the importance of the client proteins of these ER mechanisms in the plant response to the environment, it is expected that changes or alterations on their components have an impact on the plant response to environmental cues or stresses. In this mini review, we focus on the impact of the alteration of components of ERQC, ERAD and UPR in the plant response to abiotic stresses such as drought, heat, osmotic, salt and irradiation. Also, we summarize findings from recent publications looking for a connection between these processes and their possible client(s) proteins. From this, we observed that a clear connection has been established between the ERAD and UPR mechanisms, but evidence that connects ERQC components to these both processes or their possible client(s) proteins is still lacking. As a proposal, we suggest the use of proteomics approaches to uncover the identity of these proteins and their connection with ER proteostasis.

The yeast protein Ubx4p contributes to mitochondrial respiration and lithium-galactose-mediated activation of the unfolded protein response.

In the presence of galactose, lithium ions activate the unfolded protein response (UPR) by inhibiting phosphoglucomutase activity and causing the accumulation of galactose-related metabolites, including galactose-1-phosphate. These metabolites also accumulate in humans who have the disease classic galactosemia. Here, we demonstrate that Saccharomyces cerevisiae yeast strains harboring a deletion of UBX4, a gene encoding a partner of Cdc48p in the endoplasmic reticulum-associated degradation (ERAD) pathway, exhibit delayed UPR activation after lithium and galactose exposure because the deletion decreases galactose-1-phosphate levels. The delay in UPR activation did not occur in yeast strains in which key ERAD or proteasomal pathway genes had been disrupted, indicating that the ubx4Δ phenotype is ERAD-independent. We also observed that the ubx4Δ strain displays decreased oxygen consumption. The inhibition of mitochondrial respiration was sufficient to diminish galactose-1-phosphate levels and, consequently, affects UPR activation. Finally, we show that the deletion of the AMP-activated protein kinase ortholog-encoding gene SNF1 can restore the oxygen consumption rate in ubx4Δ strain, thereby reestablishing galactose metabolism, UPR activation, and cellular adaption to lithium-galactose challenge. Our results indicate a role for Ubx4p in yeast mitochondrial function and highlight that mitochondrial and endoplasmic reticulum functions are intertwined through galactose metabolism. These findings also shed new light on the mechanisms of lithium action and on the pathophysiology of galactosemia.

Multiplexed Digital mRNA Expression Analysis Profiles System-Wide Changes in mRNA Abundance and Responsiveness of UPR-Specific Gene Expression Changes During Batch Culture of Recombinant Chinese Hamster Ovary Cells.

The unfolded protein response (UPR) signaling pathway is viewed as critical for setting the effectiveness of recombinant protein expression in CHO cells. In this study, Nanostring nCounter technology is used to study expression of a group of genes associated with cellular processes linked to UPR activation under ER stress and the changing environment of a batch culture. Time course induction of ER stress, using tunicamycin (TM), shows a group of genes such as Chop, Trb3, Sqstm1, Grp78, and Herpud1 respond rapidly to TM inhibition of N-glycosylation, while others such as Atf5, Odz4, and Birc5 exhibits a delayed response. In batch culture, expression of "classical" UPR markers only increases when cells enter decline phase. In addition to providing a detailed analysis of the expression of process-relevant UPR markers during batch culture and in response to imposed chemical stress, we also highlighted six genes (Herpud1, Odz4, Sqstm1, Trb3, Syvn1, and Birc5) associated with the perception of ER stress responses in recombinant CHO cells. Herpud1 (involved in ER-associated degradation) exhibits a rapid (primary) response to stress and its relationship (and that of the other five genes) to the overall cellular UPR may identify novel targets to modulate recombinant protein production in CHO cells.

Pro-inflammatory cytokines enhance ERAD and ATF6α pathway activity in salivary glands of Sjögren's syndrome patients.

Salivary gland (SG) acinar-cells are susceptible to endoplasmic reticulum (ER) stress related to their secretory activity and the complexity of synthesized secretory products. SGs of Sjögren's syndrome patients (SS)-patients show signs of inflammation and altered proteostasis, associated with low IRE1α/XBP-1 pathway activity without avert increases in apoptosis. Acinar-cells may avoid apoptosis by activation of the ATF6α pathway and ER-associated protein degradation (ERAD). The aim of this study was to evaluate the role of pro-inflammatory cytokines in ATF6α pathway/ERAD activation and cell viability in labial salivary glands (LSG) of SS-patients. In biopsies from SS-patients increased ATF6α signaling pathway activity, as evidenced by generation of the ATF6f cleavage fragment, and increased expression of ERAD machinery components, such as EDEM1, p97, SEL1L, gp78, UBE2J1, UBE2G2, HERP and DERLIN1, were observed compared to controls. Alternatively, for pro- (active-caspase-3) and anti-apoptotic (cIAP2) markers no significant difference between the two experimental groups was detected. Increased presence of ATF6f and ERAD molecules correlated significantly with increased expression of pro-inflammatory cytokines. These observations were corroborated in vitro in 3D-acini treated with TNF-α and/or IFN-γ, where an increase in the expression and activation of the ATF6α sensor and ERAD machinery components was detected under ER stress conditions, while changes in cell viability and caspase-3 activation were not observed. Cytokine stimulation protected cells from death when co-incubated with an ERAD machinery inhibitor. Alternatively, when cytokines were eliminated from the medium prior to ERAD inhibition, cell death increased, suggesting that the presence of pro-inflammatory cytokines in the medium is essential to maintain cell viability. In conclusion, the ATF6α pathway and the ERAD machinery are active in LSG of SS-patients. Both were also activated by TNF-α and IFN-γ in vitro in 3D-acini and aided in preventing apoptosis. IFN-γ levels were elevated in SS-patients and UPR responses triggered in vitro by this cytokine closely matched those observed in LSG from SS-patients, suggesting that cytokines may induce ER stress.