Pleiotropic effects of the histone deacetylase Hos2 linked to H4‐K16 deacetylation,H3‐K56 acetylation,and H2A‐S129 phosphorylation in Beauveria bassiana

Histone acetyltransferases and deacetylases maintain dynamics of lysine acetylation/deacetylation on histones and nonhistone substrates involved in gene regulation and cellular events. Hos2 is a Class I histone deacetylases that deacetylates unique histone H4‐K16 site in yeasts. Here, we report that orthologous Hos2 deacetylates H4‐K16 and is also involved in the acetylation of histone H3‐K56 and the phosphorylation of histone H2A‐S129 and cyclin‐dependent kinase 1 CDK1‐Y15 in Beauveria bassiana, a filamentous fungal insect pathogen. These site‐specific modifications are evidenced with hyperacetylated H4‐K16, hypoacetylated H3‐K56, and both hypophosphorylated H2A‐S129 and CDK1‐Y15 in absence of hos2. Consequently, the Δhos2 mutant suffered increased sensitivities to DNA‐damaging and oxidative stresses, disturbed cell cycle, impeded cytokinesis, increased cell size or length, reduced conidiation capacity, altered conidial properties, and attenuated virulence. These phenotypic changes correlated well with dramatic repression of many genes that are essential for DNA damage repair, G₁/S transition and DNA synthesis, hyphal septation, and asexual development. The uncovered ability for Hos2 to directly deacetylate H4‐K16 and to indirectly modify H3‐K56, H2A‐S129, and CDK1‐Y15 provides novel insight into more subtle regulatory role for Hos2 in genomic stability and diverse cellular events in the fungal insect pathogen than those revealed previously in nonentomophathogenic fungi.     

The divergence and positive selection of the plant‐specific BURP‐containing protein family

BURP domain‐containing proteins belong to a plant‐specific protein family and have diverse roles in plant development and stress responses. However, our understanding about the genetic divergence patterns and evolutionary rates of these proteins remain inadequate. In this study, 15 plant genomes were explored to elucidate the genetic origins, divergence, and functions of these proteins. One hundred and twenty‐five BURP protein‐encoding genes were identified from four main plant lineages, including 13 higher plant species. The absence of BURP family genes in unicellular and multicellular algae suggests that this family (1) appeared when plants shifted from relatively stable aquatic environments to land, where conditions are more variable and stressful, and (2) is critical in the adaptation of plants to adverse environments. Promoter analysis revealed that several responsive elements to plant hormones and external environment stresses are concentrated in the promoter region of BURP protein‐encoding genes. This finding confirms that these genes influence plant stress responses. Several segmentally and tandem‐duplicated gene pairs were identified from eight plant species. Thus, in general, BURP domain‐containing genes have been subject to strong positive selection, even though these genes have conformed to different expansion models in different species. Our study also detected certain critical amino acid sites that may have contributed to functional divergence among groups or subgroups. Unexpectedly, all of the critical amino acid residues of functional divergence and positive selection were exclusively located in the C‐terminal region of the BURP domain. In conclusion, our results contribute novel insights into the genetic divergence patterns and evolutionary rates of BURP proteins.     

Disruption of gene SPL35, encoding a novel CUE domain‐containing protein,leads to cell death and enhanced disease response in rice

Lesion mimic mutants that exhibit spontaneous hypersensitive response (HR)‐like necrotic lesions are ideal experimental systems for elucidating molecular mechanisms involved in plant cell death and defence responses. Here we report identification of a rice lesion mimic mutant, spotted leaf 35 (spl35), and cloning of the causal gene by TAIL‐PCR strategy. spl35 exhibited decreased chlorophyll content, higher accumulation of H₂O₂, up‐regulated expression of defence‐related marker genes, and enhanced resistance to both fungal and bacterial pathogens of rice. The SPL35 gene encodes a novel CUE (coupling of ubiquitin conjugation to ER degradation) domain‐containing protein that is predominantly localized in cytosol, ER and unknown punctate compartment(s). SPL35 is constitutively expressed in all organs, and both overexpression and knockdown of SPL35 cause the lesion mimic phenotype. SPL35 directly interacts with the E2 protein OsUBC5a and the coatomer subunit delta proteins Delta‐COP1 and Delta‐COP2 through the CUE domain, and down‐regulation of these interacting proteins also cause development of HR‐like lesions resembling those in spl35 and activation of defence responses, indicating that SPL35 may be involved in the ubiquitination and vesicular trafficking pathways. Our findings provide insight into a role of SPL35 in regulating cell death and defence response in plants.     

NtProRP1, a novel proline‐rich protein,is an osmotic stress‐responsive factor and specifically functions in pollen tube growth and early embryogenesis in Nicotiana tabacum

Proline‐rich proteins (PRPs) are known to play important roles in sexual plant reproduction. Most of the known proteins in the family were found in styles or pollen and modulate pollen tube growth. Here, we identified a novel member of the gene family, NtProRP1, which is preferentially expressed in tobacco pollen grains, pollen tubes and zygotes. NtProRP1 could be secreted into the extracellular space including the cell wall, and the predicted N‐terminal signal peptide is crucial for its secretion. In NtProRP1‐RNAi plants, pollen germination and pollen tube growth were significantly slower and showed zigzag or swell morphology in vitro. Early embryogenesis also exhibited aberrant development, indicative of its critical role in both pollen tube growth and early embryogenesis. Further investigation revealed that NtProRP1 plays a crucial role in osmotic stress response during pollen tube growth and is likely regulated by Tsi, a stress‐responsive gene, suggesting that the regulatory mechanism is also involved in the stress response during sexual plant reproduction. These data provide evidence that NtProRP1 functions as a downstream factor of Tsi1 in the stress response and converges the stress signal into the modulation of pollen tube growth and early embryogenesis.     

The G1 phase optical reporter serves as a sensor of CDK4/6 inhibition in vivo

Visualization of cell-cycle G1 phase for monitoring the early response of cell cycle specific drug remains challenging. In this study, we developed genetically engineered bioluminescent reporters by fusing full-length cyclin E to the C-terminal luciferase (named as CycE-Luc and CycE-Luc2). Next, HeLa cell line or an ER-positive breast cancer cell line MCF-7 was transfected with these reporters. In cellular assays, the bioluminescent signal of CycE-Luc and CycE-Luc2 was accumulated in the G1 phase and decreased after exiting from the G1 phase. The expression of CycE-Luc and CycE-Luc2 fusion protein was regulated in a cell cycle-dependent manner, which was mediated by proteasome ubiquitination and degradation. Next, our in vitro and in vivo experiment confirmed that the cell cycle arrested by anti-cancer agents (palbociclib or 5-FU) was monitored quantitatively and dynamically by bioluminescent imaging of these reporters in a real-time and non-invasive manner. Thus, these optical reporters could reflect the G1 phase alternation of cell cycle, and might become a future clinically translatable approach for predicting and monitoring response to palbociclib in patients with ER-positive breast cancer.     

The isolation and identification of a secreted biomarker associated with cell stress in serum‐free CHO cell culture

One approach to improving mammalian culture productivity has been to reduce cell stress and cell death in the bioreactor, thus enhancing productivity through a longer phase of viability. Here we describe the isolation and identification of a biomarker for stress and viability loss in CHO culture. Using SELDI‐TOF mass spectrometry to profile the protein component of supernatant culture media we have identified a peak at 7.7 kDa that was associated with loss of viability toward the end of the culture and simulated stress from both toxic metabolite accumulation and nutrient depletion. The relative intensity (signal/noise ratio) of the peak increased rapidly at the onset of dropping viability toward the end of the growth phase. Also, the peak height was seen to increase significantly when cells were grown under conditions emulating ammonia accumulation and glutamine deprivation. The species has been identified as a fragment of Galectin‐1 (Gal‐1) via MS/MS fingerprinting. We propose that this peak could be utilized as a marker for early onset of stress in cell culture. This work demonstrates the efficacy of SELDI technology to identify biomarkers in mammalian cell culture and highlights its value as a tool for the monitoring and improvement of culture processes. Biotechnol. Bioeng. 2009; 104: 590–600 © 2009 Wiley Periodicals, Inc.     

Hrip1, a novel protein elicitor from necrotrophic fungus,Alternaria tenuissima,elicits cell death,expression of defence‐related genes and systemic acquired resistance in tobacco

Here, we report the identification, purification, characterization and gene cloning of a novel hypersensitive response inducing protein secreted by necrotrophic fungus, Alternaria tenuissima, designated as hypersensitive response inducing protein 1 (Hrip1). The protein caused the formation of necrotic lesions that mimic a typical hypersensitive response and apoptosis‐related events including DNA laddering. The protein‐encoding gene was cloned by rapid amplification of cDNA ends (RACE) method. The sequence analysis revealed that the cDNA is 495 bp in length and the open reading frame (ORF) encodes for a polypeptide of 163 amino acids with theoretical pI of 5.50 and molecular weight of 17 562.5 Da. Hrip1 induced calcium influx, medium alkalinization, activation of salicylic acid‐induced protein kinase and several defence‐related genes after infiltration in tobacco leaves. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, at a time when expression of defence‐related genes was activated. After several days, systemic acquired resistance was also induced. The tobacco plant cells that perceived the Hrip1 generated a cascade of signals acting at local, short, and long distances, and caused the coordinated expression of specific defence responses in a way similar to hypersensitivity to tobacco mosaic virus. Thus, Hrip1 represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance in necrotrophic fungi.     

Inhibition of deubiquitination by PR‐619 induces apoptosis and autophagy via ubi‐protein aggregation‐activated ER stress in oesophageal squamous cell carcinoma

ObjectivesTargeting the deubiquitinases (DUBs) has become a promising avenue for anti‐cancer drug development. However, the effect and mechanism of pan‐DUB inhibitor, PR‐619, on oesophageal squamous cell carcinoma (ESCC) cells remain to be investigated.Materials and MethodsThe effect of PR‐619 on ESCC cell growth and cell cycle was evaluated by CCK‐8 and PI staining. Annexin V‐FITC/PI double staining was performed to detect apoptosis. LC3 immunofluorescence and acridine orange staining were applied to examine autophagy. Intercellular Ca²⁺ concentration was monitored by Fluo‐3AM fluorescence. The accumulation of ubi‐proteins and the expression of the endoplasmic reticulum (ER) stress‐related protein and CaMKKβ‐AMPK signalling were determined by immunoblotting.ResultsPR‐619 could inhibit ESCC cell growth and induce G2/M cell cycle arrest by downregulating cyclin B1 and upregulating p21. Meanwhile, PR‐619 led to the accumulation of ubiquitylated proteins, induced ER stress and triggered apoptosis by the ATF4‐Noxa axis. Moreover, the ER stress increased cytoplasmic Ca²⁺ and then stimulated autophagy through Ca²⁺‐CaMKKβ‐AMPK signalling pathway. Ubiquitin E1 inhibitor, PYR‐41, could reduce the accumulation of ubi‐proteins and alleviate ER stress, G2/M cell cycle arrest, apoptosis and autophagy in PR‐619‐treated ESCC cells. Furthermore, blocking autophagy by chloroquine or bafilomycin A1 enhanced the cell growth inhibition effect and apoptosis induced by PR‐619.ConclusionsOur findings reveal an unrecognized mechanism for the cytotoxic effects of general DUBs inhibitor (PR‐619) and imply that targeting DUBs may be a potential anti‐ESCC strategy.     

Identification of coronin‐1a as a novel antibody target for clinically isolated syndrome and multiple sclerosis

Recently, we identified the mimotope UH‐CIS6 as a novel candidate antibody target for clinically isolated syndrome (CIS) and relapsing‐remitting (RR) multiple sclerosis (MS). The purpose of this study was to further validate UH‐CIS6 as an antibody target for CIS and MS and to identify the in vivo antibody target of UH‐CIS6. First, a UH‐CIS6 peptide ELISA was optimized. Next, we investigated the antibody response toward UH‐CIS6 in cerebrospinal fluid (CSF) from patients with CIS (n = 20), MS (n = 43) and other neurological diseases (n = 42). Immunoprecipitation of anti‐UH‐CIS6 antibodies on a normal human brain lysate was performed to identify the in vivo antibody target of UH‐CIS6. The cellular expression of an in vivo candidate target was investigated by immunohistochemistry using MS brain tissue sections. Antibody reactivity toward UH‐CIS6 was detected in a significantly increased proportion of CSF samples from CIS and RR‐MS patients as compared with neurological controls (p = 0.046). We identified and confirmed coronin‐1a as the in vivo antibody target for UH‐CIS6. Furthermore, coronin‐1a was expressed by T cells and macrophages in an active MS lesion. Together, these results demonstrate that coronin‐1a is a novel antibody target for CIS and MS.     

Isolation and characterisation of cDNA encoding a wheat heavy metal‐associated isoprenylated protein involved in stress responses

In cells, metallochaperones are important proteins that safely transport metal ions. Heavy metal‐associated isoprenylated plant proteins (HIPPs) are metallochaperones that contain a metal binding domain and a CaaX isoprenylation motif at the carboxy‐terminal end. To investigate the roles of wheat heavy metal‐associated isoprenylated plant protein (TaHIPP) genes in plant development and in stress responses, we isolated cDNA encoding the wheat TaHIPP1 gene, which contains a heavy metal‐associated domain, nuclear localisation signals and an isoprenylation motif (CaaX motif). Quantitative real‐time PCR analysis indicated that the TaHIPP1 gene was differentially expressed under biotic and abiotic stresses. Specifically, TaHIPP1 expression was up‐regulated by ABA exposure or wounding. Additionally, TaHIPP1 over‐expression in yeast (Schizosaccharomyces pombe) significantly increased the cell growth rate under Cu²⁺ and high salinity stresses. The nuclear localisation of the protein was confirmed with confocal laser scanning microscopy of epidermal onion cells after particle bombardment with chimeric TaHIPP1‐GFP constructs. In addition, TaHIPP1 was shown to enhance the susceptibility of wheat to Pst as determined by virus‐induced gene silencing. These data indicate that TaHIPP1 is an important component in defence signalling pathways and may play a crucial role in the defence response of wheat to biotic and certain abiotic stresses.