Cell Stress and Chaperones

Eukaryotic cells respond to stress caused by the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum by activating the intracellular signaling pathways referred to as the unfolded protein response (UPR). In metazoans, UPR consists of three parallel branches, each characterized by its stress sensor protein, IRE1, ATF6, and PERK, respectively. In Drosophila, IRE1/XBP1 pathway is considered to function as a major branch of UPR; however, its physiological roles during the normal development and homeostasis remain poorly understood. To visualize IRE1/XBP1 activity in fly tissues under normal physiological conditions, we modified previously reported XBP1 stress sensing systems (Souid et al., Dev Genes Evol 217: 159–167, 2007; Ryoo et al., EMBO J 26: 242-252, 2007), based on the recent reports regarding the unconventional splicing of XBP1/HAC1 mRNA (Aragon et al., Nature 457: 736–740, 2009; Yanagitani et al., Mol Cell 34: 191–200, 2009; Science 331: 586–589, 2011). The improved XBP1 stress sensing system allowed us to detect new IRE1/XBP1 activities in the brain, gut, Malpighian tubules, and trachea of third instar larvae and in the adult male reproductive organ. Specifically, in the larval brain, IRE1/XBP1 activity was detected exclusively in glia, although previous reports have largely focused on IRE1/XBP1 activity in neurons. Unexpected glial IRE1/XBP1 activity may provide us with novel insights into the brain homeostasis regulated by the UPR.

Acute pancreatitis (AP) is an inflammatory process in which cytokines and chemokines are involved. After onset, extrapancreatic stimuli can induce the expression of cytokines in pancreatic acinar cells, thereby amplifying this inflammatory loop. To further determine the role and mechanism of irritating agents in the pathogenesis of AP, rat pancreatic tissues were stimulated with ascitic fluid (APa) and serum (APs) from rats with AP or with lipopolysaccharide (LPS). In addition, the alteration of heat shock protein 60 (HSP60) expression was evaluated. Rat pancreas was removed and meticulously snipped to fragments. The snips were cultured for up to 48 h. During this period, the tissue viability as well as amylase and TNF-α levels in the supernatant and the HSP60 expression in the pancreatic tissue before and after stimulation by APa, APs, and LPS were assayed time-dependently. At different time-points during the culture, the viability and the amylase activity in the pancreatic tissue remained largely stable. After stimulation with APa, APs, or LPS for 1 h, the pancreatic tissues showed some damage, and this was followed by a sharp decrease in the viability accompanied by increased levels of amylase and TNF-α in the culture medium 2 or 4 h after stimulation (p < 0.05). In contrast, both the HSP60 mRNA and protein levels had a relatively high expression in the freshly prepared tissue fragments (0 h). As the culturing period was extended, the expression of HSP60 mRNA decreased only slightly; at the same time, the HSP60 protein levels decreased over a prolonged culture time, significantly so from 12 through 48 h (p < 0.05). After stimulation with APs, APa, or LPS, both the expression of HSP60 mRNA and protein in the tissue fragments increased slightly at 1 h and decreased significantly thereafter at 2 and 4 h (p < 0.05). APa, APs, or LPS induce injuries on isolated pancreatic tissues, accompanied by an altered HSP60 expression pattern in a time-dependent manner.

Accumulation of mis- and unfolded proteins during viral replication can cause stress in the endoplasmic reticulum (ER) and trigger the unfolded protein response (UPR). If unchecked, this process may induce cellular changes detrimental to viral replication. In the report, we investigated the impact of HSV-1 on the UPR during lytic replication. We found that HSV-1 effectively disarms the UPR in early stages of viral infection. Only ATF6 activation was detected during early infection, but with no upregulation of target chaperone proteins. Activity of the eIF2α/ATF4 signaling arm increased at the final stage of HSV-1 replication, which may indicate completion of virion assembly and egress, thus releasing suppression of the UPR. We also found that the promoter of viral ICP0 was responsive to ER stress, an apparent mimicry of cellular UPR genes. These results suggest that HSV-1 may use ICP0 as a sensor to modulate the cellular stress response.

Heat stress is exacerbated by global warming and affects human and animal health, leading to heart damage caused by imbalances in reactive oxygen species (ROS) and the antioxidant system, acid-base chemistry, electrolytes and respiratory alkalosis. Vitamin C scavenges excess ROS, and sodium bicarbonate maintains acid-base and electrolyte balance, and alleviates respiratory alkalosis. Herein, we explored the ability of vitamin C alone and in combination with equimolar sodium bicarbonate (Vitamin C-Na) to stimulate endogenous antioxidants and heat shock proteins (HSPs) to relieve heat stress in H9C2 cells. Control, vitamin C (20 μg/ml vitamin C for 16 h) and vitamin C-Na (20 μg/ml vitamin C-Na for 16 h) groups were heat-stressed for 1, 3 or 5 h. Granular and vacuolar degeneration, karyopyknosis and damage to nuclei and mitochondria were clearly reduced in treatment groups, as were apoptosis, lactate dehydrogenase activity and ROS and malondialdehyde levels, while superoxide dismutase activity was increased. Additionally, CRYAB, Hsp27, Hsp60 and Hsp70 mRNA levels were upregulated at 3 h (p < 0.01), and protein levels were increased for CRYAB at 0 h (p < 0.05) and 1 h (p < 0.01), and for Hsp70 at 3 and 5 h (p < 0.01). Thus, pre-treatment with vitamin C or vitamin C-Na might protect H9C2 cells against heat damage by enhancing the antioxidant ability and upregulating CRYAB and Hsp70.

SPL (SQUAMOSA promoter binding proteins-like) are plant-specific transcription factors that play essential roles in a variety of developmental processes as well as the ability to withstand biotic and abiotic stresses. To date, numerous species have been investigated for the SPL gene family, but so far, no SPL family genes have been thoroughly identified and characterized in the sunflower (Helianthus annuus). In this study, 25 SPL genes were identified in the sunflower genome and were unevenly distributed on 11 chromosomes. According to phylogeny analysis, 59 SPL genes from H. annuus, O. sativa, and A. thaliana were clustered into seven groups. Furthermore, the SPL genes in groups-I and II were demonstrated to be potential targets of miR156. Synteny analysis showed that 7 paralogous gene pairs exist in HaSPL genes and 26 orthologous gene pairs exist between sunflower and rice, whereas 21 orthologous gene pairs were found between sunflower and Arabidopsis. Segmental duplication appears to have played a vital role in the expansion processes of sunflower SPL genes, and because of selection pressure, all duplicated genes have undergone purifying selection. Tissue-specific gene expression analysis of the HaSBP genes proved their diverse spatiotemporal expression patterns, which were predominantly expressed in floral organs and differentially expressed in stem, axil, and root tissues. The expression pattern of HaSPL genes under water stress showed broad involvement of HaSPLs in the response to flood and drought stresses. This genome-wide identification investigation provides detailed information on the sunflower SPL transcription factor gene family and establishes a strong platform for future research on sunflower responses to abiotic stress tolerance.

Lactate produced by Sertoli cells plays an important role in spermatogenesis, and heat stress induces lactate production in immature boar Sertoli cells. Extracellular signaling regulated kinase 1 and 2 (ERK1/2) participates in heat stress response. However, the effect of ERK1/2 on heat stress-induced lactate production is unclear. In the present study, Sertoli cells were isolated from immature boar testis and cultured at 32 °C. Heat stress was induced in a 43 °C incubator for 30 min. Proteins and RNAs were detected by western blotting and RT-PCR, respectively. Lactate production and lactate dehydrogenase (LDH) activity were detected using commercial kits. Heat stress promoted ERK1/2 phosphorylation, showing a reducing trend with increasing recovery time. In addition, heat stress increased heat shock protein 70 (HSP70), glucose transporter 3 (GLUT3), and lactate dehydrogenase A (LDHA) expressions, enhanced LDH activity and lactate production at 2-h post-heat stress. Pretreatment with U0126 (1 × 10−6 mol/L), a highly selective inhibitor of ERK1/2 phosphorylation, reduced HSP70, GLUT3, and LDHA expressions and decreased LDH activity and lactate production. Meanwhile, ERK2 siRNA1 reduced the mRNA level of ERK2 and weakened ERK1/2 phosphorylation. Additionally, ERK2 siRNA1 reduced HSP70, GLUT3, and LHDA expressions decreased LDH activity and lactate production. Furthermore, HSP70 siRNA3 downregulated GLUT3 and LDHA expressions and decreased LDH activity and lactate production. These results show that activated ERK1/2 increases heat stress-induced lactate production by enhancing HSP70 expression to promote the expressions of molecules related to lactate production (GLUT3 and LDHA). Our study reveals a new insight in reducing the negative effect of heat stress in boars.

Heat shock protein 70 (HSPA) is a molecular chaperone which has been suggested to shuttle human leukocyte antigen (HLA) epitope precursors from the proteasome to the transporter associated with antigen processing. Despite the reported observations that peptides chaperoned by HSPA are an effective source of antigens for cross-priming, little is known about the peptides involved in the process. In this study, we investigated the possible involvement of HSPA in HLA class I or class II antigen presentation and analysed the antigenic potential of the associated peptides. HSPA was purified from CCRF-CEM and K562 cell lines, and using mass spectrometry techniques, we identified 44 different peptides which were co-purified with HSPA. The affinity of the identified peptides to two HSPA isoforms, HSPA1A and HSPA8, was confirmed using a peptide array. Four of the HSPA-associated peptides were matched with 13 previously reported HLA epitopes. Of these 13 peptides, nine were HLA class I and four were HLA class II epitopes. These results demonstrate the association of HSPA with HLA class I and class II epitopes, therefore providing further evidence for the involvement of HSPA in the antigen presentation process.