Heat shock and light activation of aChlamydomonas HSP70 gene are mediated by independent regulatory pathways

Induction ofHSP70 heat shock genes by light has been demonstrated inChlamydomonas. Our aim was to establish whether this induction by light is mediated by the heat stress sensing pathway or by an independent signal chain. Inhibitors of cytoplasmic protein synthesis revealed an initial difference. Cycloheximide and other inhibitors of protein synthesis preventedHSP70A induction upon illumination but not during heat stress. Analysis ofHSP70A induction in cells that had differentiated into gametes revealed a second difference. While heat shock resulted in elevatedHSP70A mRNA levels, light was no longer able to serve as an inducer in gametes. To identify the regulatory sequences that mediate the response of theHSP70A gene to either heat stress or light we introduced a series of progressive 5′ truncations into its promoter sequence. Analyses of the levels of mRNA transcribed from these deletion constructs showed that in most of them the responses to heat shock and light were similar, suggesting that light induction is mediated by a light-activated heat shock factor. However, we show that theHSP70A promoter also containscis-acting sequences involved in light induction that do not participate in induction by heat stress. Together, these results provide evidence for a regulation ofHSP70A gene expression by light through a heat shock-independent signal pathway.     

Development of rapid and highly sensitive HSPA1A promoter-driven luciferase reporter system for assessing oxidative stress associated with low-dose photodynamic therapy

Photodynamic therapy (PDT) is a regulatory-approved modality for treating a variety of malignant tumors. It induces tumor tissue damage via photosensitizer-mediated oxidative cytotoxicity. The heat shock protein 70 (HSP70-1) is a stress protein encoded by the HSPA1A gene and is significantly induced by oxidative stress associated with PDT. The aim of this study was to identify the functional region of the HSPA1A promoter that responds to PDT-induced oxidative stress and uses the stress responsiveness of HSPA1A expression to establish a rapid and cost-effective photocytotoxic assessment bioassay to evaluate the photodynamic potential of photosensitizers. By constructing luciferase vectors with a variety of hspa1a promoter fractions and examining their relative luciferase activity, we demonstrated that the DNA sequence from −218 to +87 of the HSPA1A gene could be used as a functional promoter to detect the PDT-induced oxidative stress. The maximal relative luciferase activity level of HSPA1A (HSP70-1) induced by hypericin-PDT was nearly nine times that of the control. Our results suggest that the novel reporter gene assay using a functional region of the HSP70A1A promoter has significant advantages for the detection of photoactivity in terms of both speed and sensitivity, when compared with a cell viability test based on ATP quantification and ROS levels. Furthermore, phthalocyanine zinc and methylene blue both induced significantly elevated levels of relative luciferase activity in a dose-dependent manner.     

Heat shock and light activation of aChlamydomonas HSP70 gene are mediated by independent regulatory pathways

Induction ofHSP70 heat shock genes by light has been demonstrated inChlamydomonas. Our aim was to establish whether this induction by light is mediated by the heat stress sensing pathway or by an independent signal chain. Inhibitors of cytoplasmic protein synthesis revealed an initial difference. Cycloheximide and other inhibitors of protein synthesis preventedHSP70A induction upon illumination but not during heat stress. Analysis ofHSP70A induction in cells that had differentiated into gametes revealed a second difference. While heat shock resulted in elevatedHSP70A mRNA levels, light was no longer able to serve as an inducer in gametes. To identify the regulatory sequences that mediate the response of theHSP70A gene to either heat stress or light we introduced a series of progressive 5 truncations into its promoter sequence. Analyses of the levels of mRNA transcribed from these deletion constructs showed that in most of them the responses to heat shock and light were similar, suggesting that light induction is mediated by a light-activated heat shock factor. However, we show that theHSP70A promoter also containscis-acting sequences involved in light induction that do not participate in induction by heat stress. Together, these results provide evidence for a regulation ofHSP70A gene expression by light through a heat shock-independent signal pathway.     

Analysis of Chromatin Structure in the Control Regions of the <Emphasis Type="Italic">Chlamydomonas HSP70A</Emphasis> and <Emphasis Type="Italic">RBCS2</Emphasis> Genes

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of ~700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.     

Recruitment of a Cytoplasmic Chaperone Relay by the A2A Adenosine Receptor

The adenosine A_2A receptor is a prototypical rhodopsin-like G protein-coupled receptor but has several unique structural features, in particular a long C terminus (of >120 residues) devoid of a palmitoylation site. It is known to interact with several accessory proteins other than those canonically involved in signaling. However, it is evident that many more proteins must interact with the A_2A receptor, if the trafficking trajectory of the receptor is taken into account from its site of synthesis in the endoplasmic reticulum (ER) to its disposal by the lysosome. Affinity-tagged versions of the A_2A receptor were expressed in HEK293 cells to identify interacting partners residing in the ER by a proteomics approach based on tandem affinity purification. The receptor-protein complexes were purified in quantities sufficient for analysis by mass spectrometry. We identified molecular chaperones (heat-shock proteins HSP90α and HSP70-1A) that interact with and retain partially folded A_2A receptor prior to ER exit. Complex formation between the A_2A receptor and HSP90α (but not HSP90β) and HSP70-1A was confirmed by co-affinity precipitation. HSP90 inhibitors also enhanced surface expression of the receptor in PC12 cells, which endogenously express the A_2A receptor. Finally, proteins of the HSP relay machinery (e.g. HOP/HSC70-HSP90 organizing protein and P23/HSP90 co-chaperone) were recovered in complexes with the A_2A receptor. These observations are consistent with the proposed chaperone/coat protein complex II exchange model. This posits that cytosolic HSP proteins are sequentially recruited to folding intermediates of the A_2A receptor. Release of HSP90 is required prior to recruitment of coat protein complex II components. This prevents premature ER export of partially folded receptors.     

HSP70 and Its Cochaperone CPIP Promote Potyvirus Infection in Nicotiana benthamiana by Regulating Viral Coat Protein Functions

This study demonstrates that heat shock protein 70 (HSP70) together with its cochaperone CPIP regulates the function of a potyviral coat protein (CP), which in turn can interfere with viral gene expression. HSP70 was copurified as a component of a membrane-associated viral ribonucleoprotein complex from Potato virus A–infected plants. Downregulation of HSP70 caused a CP-mediated defect associated with replication. When PVA CP was expressed in trans, it interfered with viral gene expression and replication-associated translation (RAT). However, CP produced in cis interfered specifically with RAT. CPIP binds to potyviral CP, and overexpression of CPIP was sufficient to restore RAT inhibited by expression of CP in trans. Restoration of RAT was dependent on the ability of CPIP to interact with HSP70 since expression of a J-domain mutant, CPIP^Δ66, had only a minor effect on RAT. CPIP-mediated delivery of CP to HSP70 promoted CP degradation by increasing its ubiquitination when assayed in the absence of virus infection. In conclusion, CPIP and HSP70 are crucial components of a distinct translation activity that is associated with potyvirus replication.     

Association analysis of HSP70A1A haplotypes with heat tolerance in Chinese Holstein cattle

Single-nucleotide polymorphisms (SNPs) in the coding and untranslated regions of heat shock 70 kDa protein 1A (HSP70A1A), an inducible molecular chaperone that is responsible for cellular protection against heat stress, have been reported as being associated with heat tolerance. A fragment of the HSP70A1A gene was amplified in Chinese Holstein cattle and eight novel mutations were found. We performed comprehensive linkage disequilibrium (LD) and haplotype analyses of the eight SNPs of the HSP70A1A gene and examined their involvement in heat resistance in 600 Chinese Holstein cattle. Our results revealed the presence of significant differences between individuals carrying haplotype 1 and those without haplotype 1 for most of the heat-tolerance traits. Haplotype 1 increased the risk of heat stress; however, association analysis of its combination with haplotype 2 showed the lowest rectal temperature and red blood cell K⁺ level, moderate respiratory rate, and the highest red blood cell NKA level, suggesting a heterozygote advantage in the penetration of the phenotype. Protein expression levels in white blood cells among haplotype combinations further confirmed the hypothesis that heterozygotes for haplotypes 1 and 2 are more sensitive to heat stress. We presume that these mutations may be useful in the future as molecular genetic markers to assist selection for heat tolerance in cattle.     

Potential Response to Selection of HSP70 as a Component of Innate Immunity in the Abalone Haliotis rufescens

Assessing components of the immune system may reflect disease resistance. In some invertebrates, heat shock proteins (HSPs) are immune effectors and have been described as potent activators of the innate immune response. Several diseases have become a threat to abalone farming worldwide; therefore, increasing disease resistance is considered to be a long-term goal for breeding programs. A trait will respond to selection only if it is determined partially by additive genetic variation. The aim of this study was to estimate the heritability (h ²) and the additive genetic coefficient of variation (CV _A) of HSP70 as a component of innate immunity of the abalone Haliotis rufescens, in order to assess its potential response to selection. These genetic components were estimated for the variations in the intracellular (in haemocytes) and extracellular (serum) protein levels of HSP70 in response to an immunostimulant agent in 60 full-sib families of H. rufescens. Levels of HSP70 were measured twice in the same individuals, first when they were young and again when they were pre-harvest adults, to estimate the repeatability (R), the h ² and the potential response to selection of these traits at these life stages. High HSP70 levels were observed in abalones subjected to immunostimulation in both the intracellular and extracellular haemolymph fractions. This is the first time that changes in serum levels of HSP70 have been reported in response to an immune challenge in molluscs. HSP70 levels in both fractions and at both ages showed low h ² and R, with values that were not significantly different from zero. However, HSP70 induced levels had a CV _A of 13.3–16.2% in young adults and of 2.7–8.1% in pre-harvest adults. Thus, despite its low h ², HSP70 synthesis in response to an immune challenge in red abalone has the potential to evolve through selection because of its large phenotypic variation and the presence of additive genetic variance, especially in young animals.     

Erythroid-Specific Expression of LIN28A Is Sufficient for Robust Gamma-Globin Gene and Protein Expression in Adult Erythroblasts

Increasing fetal hemoglobin (HbF) levels in adult humans remains an active area in hematologic research. Here we explored erythroid-specific LIN28A expression for its effect in regulating gamma-globin gene expression and HbF levels in cultured adult erythroblasts. For this purpose, lentiviral transduction vectors were produced with LIN28A expression driven by erythroid-specific gene promoter regions of the human KLF1 or SPTA1 genes. Transgene expression of LIN28A with a linked puromycin resistance marker was restricted to the erythroid lineage as demonstrated by selective survival of erythroid colonies (greater than 95% of all colonies). Erythroblast LIN28A over-expression (LIN28A-OE) did not significantly affect proliferation or inhibit differentiation. Greater than 70% suppression of total let-7 microRNA levels was confirmed in LIN28A-OE cells. Increases in gamma-globin mRNA and protein expression with HbF levels reaching 30–40% were achieved. These data suggest that erythroblast targeting of LIN28A expression is sufficient for increasing fetal hemoglobin expression in adult human erythroblasts.     

A novel small molecule HSP90 inhibitor,NXD30001, differentially induces heat shock proteins in nervous tissue in culture and in vivo

Heat shock proteins (HSPs) are attractive therapeutic targets for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), characterized by aberrant formation of protein aggregates. Although motor neurons have a high threshold for activation of HSP genes, HSP90 inhibitors are effective inducers. This study evaluated NXD30001, a novel, small molecule HSP90 inhibitor based on the radicicol backbone, for its ability to induce neuronal HSPs and for efficacy in an experimental model of ALS based on mutations in superoxide-dismutase 1 (SOD1). In motor neurons of dissociated murine spinal cord cultures, NXD30001-induced expression of HSP70/HSPA1 (iHSP70) and its co-chaperone HSP40/DNAJ through activation of HSF1 and exhibited a protective profile against SOD1^G93A similar to geldanamycin, but with less toxicity. Treatment prevented protein aggregation, mitochondrial fragmentation, and motor neuron death, important features of mutant SOD1 toxicity, but did not effectively prevent aberrant intracellular Ca²⁺ accumulation. NXD30001 distributed to brain and spinal cord of wild-type and SOD1^G93A transgenic mice following intraperitoneal injection; however, unlike in culture, in vivo levels of SOD1 were not reduced. NXD30001-induced expression of iHSP70 in skeletal and cardiac muscle and, to a lesser extent, in kidney, but not in liver, spinal cord, or brain, with either single or repeated administration. NXD30001 is a very useful experimental tool in culture, but these data point to the complex nature of HSP gene regulation in vivo and the necessity for early evaluation of the efficacy of novel HSP inducers in target tissues in vivo.     

HSP70iQ435A to subdue autoimmunity and support anti-tumor responses

Developing immunosuppressive therapies for autoimmune diseases comes with a caveat that immunosuppression may promote the risk of developing other conditions or diseases. We have previously shown that biolistic delivery of an expression construct encoding inducible HSP70 (HSP70i) with one amino acid modification in the dendritic cell (DC) activating moiety 435–445 (HSP70i_Q435A) to mouse skin resulted in significant immunosuppressive activity of autoimmune vitiligo, associated with fewer tissue infiltrating T cells. To prepare HSP70i_Q435A as a potential therapeutic for autoimmune vitiligo, in this study we evaluated whether and how biolistic delivery of HSP70i_Q435A in mice affects anti-tumor responses. We found that HSP70i_Q435A in fact supports anti-tumor responses in melanoma-challenged C57BL/6 mice. Biolistic delivery of the HSP70i_Q435A-encoding construct to mice elicited significant anti-HSP70 titers, and anti-HSP70 IgG and IgM antibodies recognize surface-expressed and cytoplasmic HSP70i in human and mouse melanoma cells. A peptide scan revealed that the anti-HSP70 antibodies recognize a specific C-terminal motif within the HSP70i protein. The antibodies elicited surface CD107A expression among mouse NK cells, representative of antibody-mediated cellular cytotoxicity (ADCC), supporting the concept, that HSP70i_Q435A-encoding DNA elicits a humoral response to the stress protein expressed selectively on the surface of melanoma cells. Thus, besides limiting autoimmunity and inflammation, HSP70i_Q435A elicits humoral responses that limit tumor growth and may be used in conjunction with immune checkpoint inhibitors to not only control tumor but to also limit adverse events following tumor immunotherapy.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01229-x.     

Genome-wide expression analysis of HSP70 family genes in rice and identification of a cytosolic HSP70 gene highly induced under heat stress

The heat shock protein 70 (HSP70) gene family plays a key role in protecting plant cells or tissues from thermal or oxidative stress. Although many studies have elucidated the molecular functions of individual family members, genome-wide analysis of this family is still limited, especially for crop species. Our objective was to integrate various meta-profiling data into the context of a phylogenetic tree, which would enable us to perform fine evaluation of functional dominancy or redundancy within this family. Our data indicated that a loss-of-function mutant of a rice cytosolic HSP70 gene (OsctHSP70-1) did not show a clear defective phenotype in response to high temperature because of the existence of another gene family member that was closely clustered with OsctHSP70-1 and had similar expression patterns. Moreover, the second gene showed much stronger anatomical expression. We indirectly analyzed the function of OsctHSP70-1 by studying GUS activity under the control of the endogenous promoter. We also designed a probable interaction network mediated by OsctHSP70-1 and used co-expression analysis among its components to refine the network, suggesting more probable model to explain the function of OsctHSP70-1.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Up regulation of A2B adenosine receptor on monocytes are crucially required for immune pathogenicity in Indian patients exposed to Leishmania donovani

Adenosine, an endogenous purine nucleoside is one such extracellular signalling molecule whose role in regulation of anti-inflammatory cytokines and immune pathogenicity in visceral leishmaniasis is not fully understood. Here, we investigated the relationship between Leishmania donovani infection and expression of A_2B receptor on monocytes in VL patients in their pre and post treatment stage. We also investigated the molecular mechanisms influencing the interaction between immunopathogenicity and infection by exposing Leishmania donovani pulsed macrophages to Adenosine. A direct correlation of up-regulated A_2B expression on monocytes with increased parasite load was also observed. Our results also suggested that A_2B receptor activation is critically required for the stimulatory effect of adenosine on IL-10 production and suppression of nitric oxide release. The stimulatory effect of adenosine on Leishmania donovani induced IL-10 production required ERK1/2 activation and is p-38 MAPK independent.