Expression Of Chaperones Research Articles

HSF1 Can Prevent Inflammation following Heat Shock by Inhibiting the Excessive Activation of the ATF3 and JUN&FOS Genes.

Heat Shock Factor 1 (HSF1), a transcription factor frequently overexpressed in cancer, is activated by proteotoxic agents and participates in the regulation of cellular stress response. To investigate how HSF1 level affects the response to proteotoxic stress, we integrated data from functional genomics analyses performed in MCF7 breast adenocarcinoma cells. Although the general transcriptional response to heat shock was impaired due to HSF1 deficiency (mainly chaperone expression was inhibited), a set of genes was identified, including ATF3 and certain FOS and JUN family members, whose stress-induced activation was stronger and persisted longer than in cells with normal HSF1 levels. These genes were direct HSF1 targets, suggesting a dual (activatory/suppressory) role for HSF1. Moreover, we found that heat shock-induced inflammatory response could be stronger in HSF1-deficient cells. Analyses of The Cancer Genome Atlas data indicated that higher ATF3, FOS, and FOSB expression levels correlated with low HSF1 levels in estrogen receptor-positive breast cancer, reflecting higher heat shock-induced expression of these genes in HSF1-deficient MCF7 cells observed in vitro. However, differences between the analyzed cancer types were noted in the regulation of HSF1-dependent genes, indicating the presence of cell-type-specific mechanisms. Nevertheless, our data indicate the existence of the heat shock-induced network of transcription factors (associated with the activation of TNFα signaling) which includes HSF1. Independent of its chaperone-mediated cytoprotective function, HSF1 may be involved in the regulation of this network but prevents its overactivation in some cells during stress.

Abstract P1055: Co-chaperone Bag5 Protects Cardiomyocytes From Oxidative Stress

Introduction: Cardiovascular disease is one of the leading causes of death globally. Cardiac ischemic can alter cellular homeostasis and potentially lead to heart failure. Ischemic cells modulate the expression of molecular chaperones, which assist with the folding/refolding of cellular proteins. Specifically, the heat-shock protein 70 (Hsp70) chaperone, and its co-chaperones in the Bcl-2-associated athanogene (BAG) protein family, help maintain protein homeostasis by protein folding and degradation. This project serves to explore the role of BAG5 in cardiac ischemia. Methods: Expression of BAG5 was determined in left ventricular tissue isolated from cardiomyopathy patients and in ischemic cardiomyocytes. Co-immunoprecipitation (IP) and mass spectrometry analysis were utilized to determine potential interacting partners of BAG5. For the generation of ischemic conditions, neonatal rat cardiomyocytes were incubated in a hypoxia chamber for 12 hours in DMEM media with no glucose, 0% serum, and 0.1% O2. The role of BAG5 in cellular viability, reactive oxygen species (ROS) production, and proteostasis during ischemia was determined through gain-in and knock-down approaches using adenoviruses. In addition, mitochondrial membrane potential and ROS production were determined by the TMRE and MitoSOX red staining, respectively. Mitochondrial function was determined by a Seahorse XF assay. Results: Co-immunoprecipitation and mass spectrometry analysis revealed that BAG5 interacts with Myosin and Hsp70. Expression of BAG5 in cardiomyocytes was significantly decreased during ischemia. Further, overexpression of BAG5 protects cardiomyocytes by inhibiting excessive autophagy and reducing ROS production. Moreover, BAG5 expression improves mitochondrial function during ischemia. Conclusion: Understanding the role of BAG5 within the cell may help establish the protein as a potential therapeutic molecule for the protection of the ischemic heart.

Production of Indigo by Recombinant Escherichia coli with Expression of Monooxygenase, Tryptophanase, and Molecular Chaperone.

Indigo is an important pigment widely used in industries of food, cosmetics, and textile. In this work, the styrene monooxygenase StyAB from Pseudomonas putida was co-expressed with the tryptophanase TnaA and the chaperone groES-groEL in Escherichia coli for indigo production. Over-expression of the gene styAB endowed the recombinant E. coli AB with the capacity of indigo biosynthesis from indole and tryptophan. Tryptophan fermentation in E. coli AB generated about five times more indigo than that from indole, and the maximum 530 mg/L of indigo was obtained from 1.2 mg/mL of tryptophan. The gene TnaA was then co-expressed with styAB, and the tryptophanase activity significantly increased in the recombinant E. coli ABT. However, TnaA expression led to a decrease in the activity of StyAB and indigo yield in E. coli ABT. Furthermore, the plasmid pGro7 harboring groES-groEL was introduced into E. coli AB, which obviously promoted the activity of StyAB and accelerated indigo biosynthesis in the recombinant E. coli ABP. In addition, the maximum yield of indigo was further increased to 550 mg/L from 1.2 mg/mL of tryptophan in E. coli ABP. The genetic manipulation strategy proposed in this work could provide new insights into construction of indigo biosynthesis cell factory for industrial production.

The role and mechanisms of the two-component system EnvZ/OmpR on the intracellular survival of Aeromonas hydrophila.

Aeromonas hydrophila infections are common in aquaculture. Our previous studies found that the A.hydrophila B11 strain can survive in fish macrophages for at least 24 h and the two-component system EnvZ/OmpR may be involved in intracellular survival. To reveal the role and mechanism of the two-component system EnvZ/OmpR in intracellular survival of A.hydrophila, the genes of envZ/ompR were silenced by shRNAi. The results showed that the survival rates of the envZ-RNAi and ompR-RNAi strains were only 2.05% and 3.75%, respectively, which were decreased by 91% and 83.6% compared with that of the wild-type strain. The escape ability of envZ-RNAi and ompR-RNAi was also decreased by 51.4% and 19.7%, respectively. The comparative transcriptome analysis revealed that the functional genes directly related to bacterial intracellular survival mainly included the genes related to anti-stress capacity, and the genes related to Zn2+ and Mg2+ transport. Further research confirmed that two-component system EnvZ/OmpR can regulate the expression of the important molecular chaperones, such as groEL, htpG, dnaK, clpB and grpE. The expression of these molecular chaperones in wild-type strain was up-regulated with the increase in H2 O2 concentrations, while the expression of these molecular chaperones in silent strains did not change significantly. Cells that phagocytosed wild-type strain had higher ROS content than cells that phagocytosed silent strains. Two-component system EnvZ/OmpR could also regulate zinc transporter (znuA, znuB, znuC) and zinc efflux protein (zntA) to maintain zinc homeostasis in cells, thus affecting the ability of bacteria to survive in phagocytes. Moreover, two-component system EnvZ/OmpR could affect the growth and intracellular survival of A.hydrophila by regulating the expression of MgtA, MgtC and MgtE and participating in bacterial Mg2+ homeostasis in fish macrophages.

Characteristics and Regulating Roles of Wheat TaHsfA2-13 in Abiotic Stresses.

Heat shock transcription factor (Hsf) exists widely in eukaryotes and responds to various abiotic stresses by regulating the expression of downstream transcription factors, functional enzymes, and molecular chaperones. In this study, TaHsfA2-13, a heat shock transcription factor belonging to A2 subclass, was cloned from wheat (Triticum aestivum) and its function was analyzed. TaHsfA2-13 encodes a protein containing 368 amino acids and has the basic characteristics of Hsfs. Multiple sequence alignment analysis showed that TaHsfA2-13 protein had the highest similarity with TdHsfA2c-like protein from Triticum dicoccoides, which reached 100%. The analysis of tissue expression characteristics revealed that TaHsfA2-13 was highly expressed in root, shoot, and leaf during the seedling stage of wheat. The expression of TaHsfA2-13 could be upregulated by heat stress, low temperature, H2O2, mannitol, salinity and multiple phytohormones. The TaHsfA2-13 protein was located in the nucleus under the normal growth conditions and showed a transcriptional activation activity in yeast. Further studies found that overexpression of TaHsfA2-13 in Arabidopsis thaliana Col-0 or athsfa2 mutant results in improved tolerance to heat stress, H2O2, SA and mannitol by regulating the expression of multiple heat shock protein (Hsp) genes. In summary, our study identified TaHsfA2-13 from wheat, revealed its regulatory function in varieties of abiotic stresses, and will provide a new target gene to improve stress tolerance for wheat breeding.

The plastid-encoded protein Orf2971 is required for protein translocation and chloroplast quality control.

Photosynthesis and the biosynthesis of many important metabolites occur in chloroplasts. In these semi-autonomous organelles, the chloroplast genome encodes approximately 100 proteins. The remaining chloroplast proteins, close to 3,000, are encoded by nuclear genes whose products are translated in the cytosol and imported into chloroplasts. However, there is still no consensus on the composition of the protein import machinery including its motor proteins and on how newly imported chloroplast proteins are refolded. In this study, we have examined the function of orf2971, the largest chloroplast gene of Chlamydomonas reinhardtii. The depletion of Orf2971 causes the accumulation of protein precursors, partial proteolysis and aggregation of proteins, increased expression of chaperones and proteases, and autophagy. Orf2971 interacts with the TIC (translocon at the inner chloroplast envelope) complex, catalyzes ATP (adenosine triphosphate) hydrolysis, and associates with chaperones and chaperonins. We propose that Orf2971 is intimately connected to the protein import machinery and plays an important role in chloroplast protein quality control.

Abstract 6065: ASF1B potentiates stem cell traits and tumor progression in hepatocellular carcinoma via histone H3.3-dependent transcriptional reprogramming

Abstract Hepatocellular carcinoma (HCC) is one of the frequently diagnosed cancers worldwide and has emerged as the main cause of cancer-related death with a very poor prognosis, which is closely related to cancer stem cell (CSC), a subset of cells known to be a root of tumor recurrence, metastasis, and chemoresistance in HCC. However, the characteristics of CSCs in hepatocellular carcinoma (HCC) are still elusive. Our previous in vitro hepatocyte differentiation model revealed that the expression of anti-silencing function 1B histone chaperone (ASF1B) was remarkably upregulated in the stem cell-like stages. In HCC patients, ASF1B was highly expressed in cancer tissues compared with normal counterparts and was positively correlated with poor survival. Functional assays indicated that ASF1B could considerably promote the self-renewal, growth, and metastasis of HCC both in vitro and in vivo. Mechanistically, ASF1B could directly bind to histone H3 Variant H3.3 and enhance its incorporation into the promoter regions of certain stemness- and metastasis-associated genes including SOX9, KLF4, and TBX3, contributing to their augmented transcriptional and protein expression levels. H3.3 knock-down decreased the expression of these stemness and metastasis-associated genes, eliminating the stemness and oncogenic properties of ASF1B. Overall, our study revealed that ASF1B could facilitate HCC stemness and progression via H3.3-dependent transcriptional reprogramming. ASF1B might be a novel diagnostic and prognostic biomarker for HCC patients. Citation Format: Xiaona Fang, Beilei Liu, Shan Liu, Yu Zhang, Lanqi Gong, Qian Yan, Xinyuan Guan. ASF1B potentiates stem cell traits and tumor progression in hepatocellular carcinoma via histone H3.3-dependent transcriptional reprogramming [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6065.

Abstract 5684: Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress

Abstract Cancer cells survive under extreme environments that alter protein homeostasis, thereby provoking cellular stress responses, such as the unfolded protein response (UPR) and the integrated stress response (ISR). These pathways increase the expression of molecular chaperones to prevent the accumulation of toxic proteins in the cell. One molecular chaperone in particular, Hsp70, plays a central role in both cellular protection and the response to proteotoxic insults. This is perhaps most evident in cancer cells, some of which overexpress Hsp70 and thrive even when harboring high levels of misfolded, aggregated, and/or unassembled protein complexes. In an effort to define how cells compensate for compromised proteostasis, we examined the adaptive response in breast cancer cells after challenge with a specific Hsp70 inhibitor. We first showed that established breast cancer cell lines bin into classes based on their sensitivity to the drug. We next discovered that resistant cells have higher autophagy levels compared to the more sensitive lines. Moreover, gene and protein expression studies and imaging methods showed that autophagy was further induced by Hsp70 inhibition in resistant breast cancer cells. Consistent with the fact that Hsp70 normally targets aberrant proteins for degradation, our data indicated that resistance to compromised Hsp70 activity arose from elevated levels of autophagy. We next showed that Hsp70 inhibition specifically triggers the ISR pathway. Unexpectedly, protection against the Hsp70 inhibitor was dependent on the ISR sensor, GCN2, which is most commonly associated with the amino acid starvation response. In contrast, sensitive cells succumbed to Hsp70 inhibition by activating the PERK pathway. These data reveal an unexpected route through which cancer cells can adapt to chaperone targeted-proteotoxicity and position GCN2 and autophagy as critical compensatory mechanisms for cancer cells when proteostasis is compromised. Citation Format: Sara Sannino, Jeffrey L. Brodsky. Protein homeostasis modulation is critical for cancer cell survival upon chaperone mediated proteotoxic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5684.

HLH-1 Modulates Muscle Proteostasis During Caenorhabditis elegans Larval Development.

Muscle proteostasis is shaped by the myogenic transcription factor MyoD which regulates the expression of chaperones during muscle differentiation. Whether MyoD can also modulate chaperone expression in terminally differentiated muscle cells remains open. Here we utilized a temperature-sensitive (ts) conditional knockdown nonsense mutation in MyoD ortholog in C. elegans, HLH-1, to ask whether MyoD plays a role in maintaining muscle proteostasis post myogenesis. We showed that hlh-1 is expressed during larval development and that hlh-1 knockdown at the first, second, or third larval stages resulted in severe defects in motility and muscle organization. Motility defects and myofilament organization were rescued when the clearance of hlh-1(ts) mRNA was inhibited, and hlh-1 mRNA levels were restored. Moreover, hlh-1 knockdown modulated the expression of chaperones with putative HLH-1 binding sites in their promoters, supporting HLH-1 role in muscle maintenance during larval development. Finally, mild disruption of hlh-1 expression during development resulted in earlier dysregulation of muscle maintenance and function during adulthood. We propose that the differentiation transcription factor, HLH-1, contributes to muscle maintenance and regulates cell-specific chaperone expression post differentiation. HLH-1 may thus impact muscle proteostasis and potentially the onset and manifestation of sarcopenia.

Chaperone networks are shaped by cellular differentiation and identity.

Chaperone expression is developmentally regulated, establishing tissue-specific networks. However, the molecular basis underlying this specificity is mainly unknown. Recent evidence suggests that chaperone network rewiring is mediated, in part, by differentiation transcription factors to fit the proteome folding demands, with implications for the tissue-specific manifestation of protein misfolding diseases.

200-OR: IER3IP1 Is Critical for Maintaining Glucose Homeostasis through Regulating the Endoplasmic Reticulum Function and Survival of ß-Cells

Recessive mutations in IER3IP1 (Immediate Early Response 3 Interacting Protein 1) cause a triad of microcephaly, epilepsy, and permanent neonatal diabetes syndrome (MEDS) . IER3IP1 encodes an endoplasmic reticulum (ER) membrane protein, which is crucial for brain development, however the role of IER3IP1 in β cell function and glucose homeostasis remains unknown. Herein, we established a new β cell-specific IER3IP1 knockout (IER3IP1-βKO) mouse model and found that IER3IP1 deficiency in β cells causes severe early onset insulin deficient diabetes. Functional studies revealed a markedly dilated ER along with significantly increased proinsulin misfolding and elevated expression of the ER chaperones including PDI, ERO1, BiP, and P58IPK. Transcriptome analysis confirmed by qRT-PCR revealed a markedly decreased expression of genes associated with β cell maturation, cell cycle, and anti-apoptosis, whereas there was a significantly increased expression of anti-proliferation genes. Indeed, multiple independent approaches further demonstrated that IER3IP1-βKO impaired β cell maturation and proliferation, and promoted cell death. These data not only uncover a critical role of IER3IP1 in normal β cell function and survival, but they also provide insight into the mechanism (s) by which loss-of-function mutations of IER3IP1 lead to diabetes. Disclosure J.Yang: None. J.Sun: None. N.Li: None. T.Liu: None. S.Wang: None. X.Zhang: None. P.Arvan: n/a. M.Liu: None. J.Zhen: None. W.Feng: None. Z.Fan: None. Y.Huang: None. H.Shu: None. X.Li: None. J.Qiao: None. Y.Fan: None. Funding National Natural Science Foundation of China (81830025, 81620108004, 81870533, 81900720, 81800733, 82100865, 82070805, and 81870535)

Understanding the bug that make our drugs: Cellular responses to therapeutic protein secretion in E. coli

Production of therapeutic proteins is a $200 billion industry with E. coli being one of the main expression systems used and proteins often being secreted to the periplasm to produce disulphide bonds. However, little is known about how E. coliresponds to secretion of these therapeutic proteins into the periplasm under industrial conditions. Proteomics and transcriptomics were conducted on cultures grown at high cell density in industrial production conditions to look at the effect of secreting a model therapeutic protein (scFv) to the periplasm compared to expressing the same protein in the cytoplasm. 82 proteins and 1653 transcripts were differentially expressed when overexpressing the scFv in different cellular compartments. ScFv secretion had a significant effect on expression of genes involved in the envelope stress response. However, while certain envelope stress pathways where upregulated upon secretion others were downregulated (including expression of some periplasmic chaperones). Scfv secretion also led to an increase in expression of genes involved in degradation of membrane proteins and the SEC secretion system, as well as iron transport, Outer membrane porins and flagella operons. In future and current experiments, we are testing whether knocking out and overexpressing a selection of these genes improves secretion and folding of scFvs and other recombinant proteins.

Sequestration of TDP-43216-414 Aggregates by Cytoplasmic Expression of the proSAAS Chaperone.

As neurons age, protein homeostasis becomes less efficient, resulting in misfolding and aggregation. Chaperone proteins perform vital functions in the maintenance of cellular proteostasis, and chaperone-based therapies that promote sequestration of toxic aggregates may prove useful in blocking the development of neurodegenerative disease. We previously demonstrated that proSAAS, a small secreted neuronal protein, exhibits potent chaperone activity against protein aggregation in vitro and blocks the cytotoxic effects of amyloid and synuclein oligomers in cell culture systems. We now examine whether cytoplasmic expression of proSAAS results in interactions with protein aggregates in this cellular compartment. We report that expression of proSAAS within the cytoplasm generates dense, membraneless 2 μm proSAAS spheres which progressively fuse to form larger spheres, suggesting liquid droplet-like properties. ProSAAS spheres selectively accumulate a C-terminally truncated fluorescently tagged form of TDP-43, initiating its cellular redistribution; these TDP-43-containing spheres also exhibit dynamic fusion. Efficient encapsulation of TDP-43 into proSAAS spheres is driven by its C-terminal prion-like domain; spheres must be formed for sequestration to occur. Three proSAAS sequences, a predicted coiled-coil, a conserved region (residues 158-169), and the positively charged sequence 181-185, are all required for proSAAS to form spheres able to encapsulate TDP-43 aggregates. Substitution of lysines for arginines in the 181-185 sequence results in nuclear translocation of proSAAS and encapsulation of nuclear-localized TDP-43216-414. As a functional output, we demonstrate that proSAAS expression results in cytoprotection against full-length TDP-43 toxicity in yeast. We conclude that proSAAS can act as a functional holdase for TDP-43 via this phase-separation property, representing a cytoprotectant whose unusual biochemical properties can potentially be exploited in the design of therapeutic molecules.

Zinc Depletion Inhibits the Synthesis and Secretion of Thyroglobulin by Inducing Endoplasmic Reticulum Stress in PCCL3 Thyroid Cells

Thyroglobulin (Tg) is essential for thyroid hormone synthesis and thyroid function. Its levels are regulated by external environmental changes. Zinc is widely involved in cellular processes as a cofactor of enzymes and participates in metabolic processes. Here we investigated whether zinc depletion affected Tg production and secretion through the endoplasmic reticulum (ER) in the PCCL3 thyroid cell line exposed to the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (Tpen). Although zinc depletion did not affect the gene expression of ER chaperones (BiP and PDI), it increased the expression of ER transmembrane signaling proteins (PKR-like ER kinase, inositol requiring enzyme 1, and activating transcription factor 6 (ATF6)). This resulted in the activation of downstream factors as shown by the increase of eIF2-α phosphorylation, X-box binding protein 1 mRNA splicing, and ATF6 fragmentation. Zinc depletion induced an inhibition of Tg expression and secretion and activated apoptosis in PCCL3 cells. Moreover, a reduction of secreted T4 levels and histologically abnormal thyroid follicle structures were found after zinc depletion. Therefore, zinc depletion likely inhibited the biosynthesis and extracellular secretion of Tg through ER stress signaling. These findings provide valuable insight into zinc potential as a treatment of hyperthyroidism.

Role of HSP90 Family Molecular Chaperone in Regulating Chloroplast Protein Homeostasis and Plant Development in Arabidopsis

Chloroplasts carry out photosynthesis in plants and provide most food sources for living organisms. Either divided or differentiated from proplastids and etioplasts, the biogenesis of thylakoid membranes is the hallmark of chloroplast maturation and functionality, and this process requires a large set of photosynthetic proteins that must be synthesized on cytosolic ribosomes and then imported into the chloroplast stroma. Thylakoid lumen proteins travel further through the stroma and then cross the thylakoid membrane through one of the ancestral translocases. Like the cytosolic environment and the other major organelles, a cohort of molecular chaperones and proteases reside within the chloroplast to ensure proper protein homeostasis. We have identified and confirmed the interaction between the essential plastid HSP90 family heat shock protein HSP90C and a thylakoid lumen located photosystem II subunit PsbO1. To understand the biological significance of the interaction, we adopted transgenic approaches, specific HSP90 inhibitors, biochemical and in organello protein transport assays to study how the HSP90 family protein facilitates the representative lumen proteins transport across the thylakoid membrane. Specifically, we showed that the expression of the molecular chaperone is developmentally regulated, and its expression is highly correlated with some photosynthetic proteins while not with the others. Additionally, we demonstrated that HSP90C physically interacts with the thylakoid SEC translocon subunit, and that its chaperone activity is required for efficient PsbO1 transport. We also investigated the signature sequences of different HSP90 subfamily proteins and provided evidence that the chloroplast located HSP90C contains features specialized for some photosynthetic proteins interaction, and thus the chloroplast maturation and functionality.

The PERKs of mitochondria protection during stress: insights for PERK modulation in neurodegenerative and metabolic diseases.

Protein kinase RNA-like ER kinase (PERK) is an endoplasmic reticulum (ER) stress sensor that responds to the accumulation of misfolded proteins. Once activated, PERK initiates signalling pathways that halt general protein production, increase the efficiency of ER quality control, and maintain redox homeostasis. PERK activation also protects mitochondrial homeostasis during stress. The location of PERK at the contact sites between the ER and the mitochondria creates a PERK-mitochondria axis that allows PERK to detect stress in both organelles, adapt their functions and prevent apoptosis. During ER stress, PERK activation triggers mitochondrial hyperfusion, preventing premature apoptotic fragmentation of the mitochondria. PERK activation also increases the formation of mitochondrial cristae and the assembly of respiratory supercomplexes, enhancing cellular ATP-generating capacity. PERK strengthens mitochondrial quality control during stress by promoting the expression of mitochondrial chaperones and proteases and by increasing mitochondrial biogenesis and mitophagy, resulting in renewal of the mitochondrial network. But how does PERK mediate all these changes in mitochondrial homeostasis? In addition to the classic PERK-eukaryotic translation initiation factor 2α (eIF2α)-activating transcription factor 4 (ATF4) pathway, PERK can activate other protective pathways - PERK-O-linked N-acetyl-glucosamine transferase (OGT), PERK-transcription factor EB (TFEB), and PERK-nuclear factor erythroid 2-related factor 2 (NRF2) - contributing to broader regulation of mitochondrial dynamics, metabolism, and quality control. The pharmacological activation of PERK is protective in models of neurodegenerative and metabolic diseases, such as Huntington's disease, progressive supranuclear palsy and obesity, while the inhibition of PERK was protective in models of Parkinson's and prion diseases and diabetes. In this review, we address the molecular mechanisms by which PERK regulates mitochondrial dynamics, metabolism and quality control, and discuss the therapeutic potential of targeting PERK in neurodegenerative and metabolic diseases.

Effect of the endoplasmic reticulum stressor tunicamycin in Angomonas deanei heat-shock protein expression and on the association with the endosymbiotic bacterium

The endoplasmic reticulum (ER) presents unique properties to establishing bacterium symbiosis in eukaryotic cells since it synthesizes and glycosylates essential molecules like proteins and lipids. Tunicamycin (TM) is an antibiotic that inhibits the first step in the N-linked glycosylation in eukaryotes and has been used as an ER stress inducer to activate the Unfolded Protein Response (UPR). Mutualistic symbiosis in trypanosomatids is characterized by structural adaptations and intense metabolic exchanges, thus we investigated the effects of TM in the association between Angomonas deanei and its symbiotic bacterium, through ultrastructural and proteomic approaches. Cells treated with the inhibitor showed a decrease in proliferation, enlargement of the ER and Golgi cisternae and an increased distance between the symbiont and the ER. TM proved to be an important tool to better understand ER stress in trypanosomatids, since changes in protein composition were observed in the host protozoan, especially the expression of the Hsp90 chaperone. Furthermore, data obtained indicates the importance of the ER for the adaptation and maintenance of symbiotic associations between prokaryotes and eukaryotes, considering that this organelle has recognized importance in the biogenesis and division of cell structures.

Alterations in Proteostasis System Components in Peripheral Blood Mononuclear Cells in Parkinson Disease: Focusing on the HSP70 and p62 Levels.

Parkinson disease (PD) is attributed to a proteostasis disorder mediated by α-synuclein accumulating in a specific brain region. PD manifestation is often related to extraneuronal alterations, some of which could be used as diagnostic or prognostic PD biomarkers. In this work, we studied the shifts in the expression of proteostasis-associated chaperones of the HSP70 family and autophagy-dependent p62 protein values in the peripheral blood mononuclear cells (PBMC) of mild to moderate PD patients. Although we did not detect any changes in the intracellular HSP70 protein pool in PD patients compared to non-PD controls, an increase in the transcriptional activity of the stress-associated HSPA1A/B and HSPA6 genes was observed in these cells. Basal p62 content was found to be increased in PD patients’ PBMC, similarly to the p62 level in substantia nigra neural cells in PD. Moreover, the spontaneous apoptosis level was increased among PBMC and positively correlated with the p62 intracellular level in the PD group. A combined HSPA6- and p62-based analysis among 26 PD patients and 36 age-matched non-PD controls pointed out the diagnostic significance of these markers, with intermediate sensitivity and high specificity of this combination when observing patients diagnosed with PD.

The Identity of the Constriction Region of the Ribosomal Exit Tunnel Is Important to Maintain Gene Expression in Escherichia coli.

ABSTRACTMutational changes in bacterial ribosomes often affect gene expression and consequently cellular fitness. Understanding how mutant ribosomes disrupt global gene expression is critical to determining key genetic factors that affect bacterial survival. Here, we describe gene expression and phenotypic changes presented in Escherichia coli cells carrying an uL22(K90D) mutant ribosomal protein, which displayed alterations during growth. Ribosome profiling analyses revealed reduced expression of operons involved in catabolism, indole production, and lysine-dependent acid resistance. In general, translation initiation of proximal genes in several of these affected operons was substantially reduced. These reductions in expression were accompanied by increases in the expression of acid-induced membrane proteins and chaperones, the glutamate-decarboxylase regulon, and the autoinducer-2 metabolic regulon. In agreement with these changes, uL22(K90D) mutant cells had higher glutamate decarboxylase activity, survived better in extremely acidic conditions, and generated more biofilm in static cultures compared to their parental strain. Our work demonstrates that a single mutation in a non-conserved residue of a ribosomal protein affects a substantial number of genes to alter pH resistance and the formation of biofilms.IMPORTANCE All newly synthesized proteins must pass through a channel in the ribosome named the exit tunnel before emerging into the cytoplasm, membrane, and other compartments. The structural characteristics of the tunnel could govern protein folding and gene expression in a species-specific manner but how the identity of tunnel elements influences gene expression is less well-understood. Our global transcriptomics and translatome profiling demonstrate that a single substitution in a non-conserved amino acid of the E. coli tunnel protein uL22 has a profound impact on catabolism, cellular signaling, and acid resistance systems. Consequently, cells bearing the uL22 mutant ribosomes had an increased ability to survive acidic conditions and form biofilms. This work reveals a previously unrecognized link between tunnel identity and bacterial stress adaptation involving pH response and biofilm formation.

Canine osteosarcoma cells exhibit basal accumulation of multiple chaperone proteins and are sensitive to small molecule inhibitors of GRP78 and heat shock protein function.

Osteosarcoma is the most common type of bone cancer in dogs and humans, with significant numbers of patients experiencing treatment failure and disease progression. In our search for new approaches to treat osteosarcoma, we previously detected multiple chaperone proteins in the surface-exposed proteome of canine osteosarcoma cells. In the present study, we characterized expression of representative chaperones and find evidence for stress adaptation in canine osteosarcoma cells relative to osteogenic progenitors from normal bone. We compared the cytotoxic potential of direct (HA15) and putative (OSU-03012) inhibitors of Grp78 function and found canine POS and HMPOS osteosarcoma cells to be more sensitive to both compounds than normal cells. HA15 and OSU-03012 increased the thermal stability of Grp78 in intact POS cells at low micromolar concentrations, but each induced distinct patterns in Grp78 expression without significant change in Grp94. Both inhibitors were as effective alone as carboplatin and showed little evidence of synergy in combination treatment. However, HMPOS cells with acquired resistance to carboplatin were sensitive to inhibition of Grp78 (by HA15; OSU-03012), Hsp70 (by VER-155008), and Hsp90 (by 17-AAG) function. These results suggest that multiple nodes within the osteosarcoma chaperome may be relevant chemotherapeutic targets against platinum resistance.