2A Protein Research Articles

The role of protein p16INK4a in non-oropharyngeal head and neck squamous cell carcinoma in Southern China.

Cyclin-dependent kinase inhibitor 2A (p16INK4a) protein is a surrogate immunohistochemical marker of human papillomavirus infection in oropharynx squamous cell carcinoma (OPSCC). However, the effects of p16INK4a in non-OPSCC require additional analysis. In addition, major gaps remain in the literature, including small volumes of data for China. Therefore, the present study evaluated the frequency of p16INK4a positivity in patients with non-OPSCC in Southern China, and assessed its prognostic value. p16INK4a expression status in patients with non-OPSCC was determined by immunohistochemistry. p16INK4a-positive expression was defined as a strong and diffuse staining in ≥70% of the tumor cells. Then, the diagnostic value of p16INK4a in predicting overall survival (OS) and disease-free survival (DFS) rate was determined. The positive rate of p16INK4a was 26.3% in larynx cancer and 24.8% in oral cavity cancer. Multivariate analysis revealed that the protein status independently predicted improved OS rate, but not DFS rate (P=0.096). Comparing different disease stages, patients at an early stage with p16INK4a-positive non-OPSCC exhibited improved DFS and OS rates compared with those exhibited by patients who were negative. The p16INK4a-positive rate in patients with non-OPSCC was 25.1% [26.3% in Laryngeal squamous cell carcinoma (LSCC) and 24.8% in Oropharyngeal squamous cell carcinomas (OSCC)] in the present cohort from South China. The present study suggested that p16INK4a expression in non-OPSCC predicts favorable clinical outcomes, particularly in early stage non-OPSCC and oral cancer.

Evidence for Multiple Applications of Monoclonal Antibody 5G10.

We had earlier obtained a murine monoclonal antibody (mAb), termed 5G10, that bound to Salmonella flagellin (SF) and subsequently impaired the latter property of Toll-like receptor 5 (TLR5) signaling activation. Besides interrupting SF-mediated TLR5 activation, mAb 5G10 probably had other potential applications. In this study, we explored multiple functions of 5G10. A short peptide QRVRELAV (designated T5) derived from SF in either terminal of proteins was specifically recognized by 5G10. T5 tag expressed in eukaryotic cell was also detected by 5G10 when analyzed by Western blot, immunofluorescence assay (IFA), and fluorescent-activated cell sorting (FACS). The result of the co-immunoprecipitation assay showed that 5G10 as a bait antibody dragged out the complex of enterovirus 71 (EV71) 2A and mitochondrial antiviral signaling (MAVS) protein. More importantly, 5G10 helped to purify fusion proteins T5-tagged (EV71) 2A and T5-Japanese encephalitis virus NS5 methyltransferase (MTase). Thus, it has been suggested that mAb 5G10 could be useful in several biological applications, including protein identification, location, and affinity purification.

Protective effect of curcumin on dopamine neurons in Parkinson's disease and its mechanism

To investigate the effect of curcumin on dopamine neurons in Parkinson's disease (PD) and its mechanism. SH-SY5Y human neuroblastoma cells were treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to establish the PD cell model. The model cells were treated with curcumin and/or autophagy inhibitor 3-MA. After 48 h of drug treatment, the number of surviving dopamine neurons was detected by tyrosine hydroxylase immunofluorescence method. Western blotting was used to detect protein expression of α-Synuclein (α-Syn), transcription factor EB (TFEB) and autophagy-related proteins lysosome-associated membrane protein 2A (LAMP2A) and microtubule-associated protein 1 light chain 3-Ⅱ(LC3-Ⅱ); RT-PCR was used to detect mRNA expression of α-Syn. Compared with MPTP model group, curcumin increased the number of surviving dopamine neurons(P<0.01), decreased both protein expression and mRNA expression of α-Syn (all P<0.01), and increased protein expression of TFEB, LAMP2A and LC3-Ⅱ (all P<0.01). When curcumin and 3-MA were given concurrently, the number of surviving dopamine neurons, protein expression of TFEB, LAMP2A and LC3-Ⅱ increased (P<0.05 or P<0.01), and both protein expression and mRNA expression of α-Syn decreased (P<0.05 or P<0.01) compared with MPTP model group; but the number of surviving dopamine neurons and protein expression of LAMP2A and LC3-Ⅱ decreased compared with curcumin group (all P<0.05). Curcumin exerts protective effect on dopamine neurons in PD, which may be associated with enhancing autophagy and promoting the clearance of α-Syn.

Identification of two integration sites in favor of transgene expression in Trichoderma reesei

BackgroundThe ascomycete fungus Trichoderma reesei was widely used as a biotechnological workhorse for production of cellulases and recombinant proteins due to its large capacity of protein secretion. Transgenesis by random integration of a gene of interest (GOI) into the genome of T. reesei can generate series of strains that express different levels of the indicated transgene. The insertion site of the GOI plays an important role in the ultimate production of the targeted proteins. However, so far no systematic studies have been made to identify transgene integration loci for optimal expression of the GOI in T. reesei. Currently, only the locus of exocellobiohydrolases I encoding gene (cbh1) is widely used as a promising integration site to lead to high expression level of the GOI. No additional sites associated with efficient gene expression have been characterized.ResultsTo search for gene integration sites that benefit for the secreted expression of GOI, the food-and-mouth disease virus 2A protein was applied for co-expression of an Aspergillus niger lipA gene and Discosoma sp. DsRed1 gene in T. reesei, by random integration of the expression cassette into the genome. We demonstrated that the fluorescent intensity of RFP (red fluorescent protein) inside of the cell was well correlated with the secreted lipase yields, based on which, we successfully developed a high-throughput screening method to screen strains with relatively higher secreted expression of the GOI (in this study, lipase). The copy number and the insertion sites of the transgene were investigated among the selected highly expressed strains. Eventually, in addition to cbh1 gene locus, two other genome insertion loci that efficiently facilitate gene expression in T. reesei were identified.ConclusionsWe have successfully developed a high-throughput screening method to screen strains with optimal expression of the indicated secreted proteins in T. reesei. Moreover, we identified two optimal genome loci for transgene expression, which could provide new approach to modulate gene expression levels while retaining the indicated promoter and culture conditions.

Generating Neutralizing Antibody responses using Recombinant Infectious Rhinovirus

Abstract Human Rhinovirus (RV), a leading causative agent for common colds and the subsequent development of pneumonia, can exacerbate both asthma and chronic obstructive pulmonary disease (COPD). There is no vaccine against RV yet. As previous studies demonstrated that RV-neutralizing antibodies (nAb) correlates with protection, eliciting robust nAb responses might be a key factor for ideal RV vaccine development. Five neutralizing immunogenic (NIm) sites were identified. NIm-IA, IB, and IV are located on VP1, while NIm-II and III are located on VP2 and VP3, respectively. Since the capsid proteins (VP1, 2, 3 and 4) seem to be immunogenic, we hypothesize that a chimeric virus expressing the capsid proteins is sufficient to elicit nAb responses. The HRV-A76 insert expressing the four capsid proteins and 2A protein was cloned into a bacterial artificial chromosome (BAC) harboring cDNA expressing the full-length genome of HRV-A33. Utilizing reverse genetics, a capsid-chimeric recombinant RV strain, BAC HRV33cap76, was generated using the four capsid proteins and 2A protein from HRV-A76, and the remaining part of the genome from HRV-A33. Following RNA transfection and recovery, we found that the titer of BAC HRV33cap76 was similar to biological HRV33 or HRV76. We next immunized BALB/c mice intramuscularly with inactivated BAC HRV33cap76 mixed with alum adjuvant to determine its immunogenicity. The BAC HRV33cap76 immunization resulted in nAb against only HRV-A76. Our findings demonstrated that the capsid proteins are responsible for the generation of nAb against RV. As a capsid-chimeric recombinant RV was capable of eliciting robust nAb, it could be a novel vaccine platform to generate highly effective and safe vaccine for preventing RV infection.

Arabidopsis Homologues to the LRAT a Possible Substrate for New Plant-Based Anti-Cancer Drug Development

This article describes how an NC gene family has been identified in the genome of the Arabidopsis thaliana (Arabidopsis) by homology to the human Lecithin Retinal Acyl Transferase (LRAT) and the picornavirus 2A protein. The Arabidopsis proteins contain two motifs identified in a vast variety of organisms, an H-Box and an NC. Among related proteins are the C. elegans EGL-26, a regulator protein of cell morphogenesis in the vulva region, and human proteins that might be related to cell proliferation or development. Human homologues include HRAS-like tumour suppressors, the Tazarotene-induced gene 3 (TIG3), and a deSumoylating Isopeptidase (PNAS-4) that induces apoptosis in lung cancer cells. Preservation of the two motifs observed in the Arabidopsis proteins in homology to tumour suppressors, and the conservation of residues important for the function of the LRAT amongst the Arabidopsis homologues can be indicative not only of the importance of these domains for the function of the plant proteins but can also reveal a new candidate group for the design of plant-based tumour-targeting drug development.

β-Adrenergic regulation of cardiac type 2A protein phosphatase through phosphorylation of regulatory subunit B56δ at S573

BackgroundType 2A protein phosphatase (PP2A) enzymes are serine/threonine phosphatases which comprise a scaffold A subunit, a regulatory B subunit and a catalytic C subunit, and have been implicated in the dephosphorylation of multiple cardiac phosphoproteins. B subunits determine subcellular targeting, substrate specificity and catalytic activity, and can themselves be regulated by post-translational modifications. We explored potential β-adrenergic regulation of PP2A in cardiomyocytes through phosphorylation of the regulatory B subunit isoform B56δ. Methods and resultsPhosphate affinity SDS-PAGE and immunoblot analysis revealed increased phosphorylation of B56δ in adult rat ventricular myocytes (ARVM) exposed to the β-adrenergic receptor (βAR) agonist isoprenaline (ISO). Phosphorylation of B56δ occurred at S573, primarily through stimulation of the β1AR subtype, and was dependent on PKA activity. The functional role of the phosphorylation was explored in ARVM transduced with adenoviruses expressing wild type (WT) or non-phosphorylatable (S573A) B56δ, fused to GFP at the N-terminus. C subunit expression was increased in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A, both of which co-immunoprecipitated with endogenous C and A subunits. PP2A activity in cell lysates was increased in response to ISO in ARVM expressing GFP-B56δ-WT but not GFP-B56δ-S573A. Immunoblot analysis of the phosphoproteome in ARVM expressing GFP-B56δ-WT or GFP-B56δ-S573A with antibodies detecting (i) phospho-serine/threonine residues in distinct kinase substrate motifs or (ii) specific phosphorylated residues of functional importance in selected proteins revealed a comparable phosphorylation profile in the absence or presence of ISO stimulation. ConclusionsIn cardiomyocytes, βAR stimulation induces PKA-mediated phosphorylation of the PP2A regulatory subunit isoform B56δ at S573, which increases associated PP2A catalytic activity. This is likely to regulate the phosphorylation status of specific B56δ-PP2A substrates, which remain to be identified.

Genomic analysis of a novel picornavirus from a migratory waterfowl, greater white-fronted goose (Anser albifrons).

The complete genome of goose picornavirus 1 (GPV-1) strain goose/NLSZK2/HUN/2013 (MF358731) was determined by RT-PCR and next-generation sequencing from a cloacal sample of a migratory waterfowl, greater white-fronted goose (Anser albifrons) in Hungary. The genome of GPV-1 shows an L-3-3-4 organization pattern with a 5'-terminal origin of replication (ORI) region, a type-IV IRES, and an Hbox/NC-type 2A protein. This virus showed the highest overall sequence identity to the members of the genus Kobuvirus, although the phylogenetic position of GPV-1 is different in the analyzed P1, 2C and 3CD phylogenetic trees, which further increases the diversity of known avian picornaviruses.

Novel fluorochromes label tonoplast in living plant cells and reveal changes in vacuolar organization after treatment with protein phosphatase inhibitors.

The recently synthesized isocyanonaphtalene derivatives ACAIN and CACAIN are fluorochromes excitable at wavelengths of around 366nm and bind cysteine-rich proteins with hydrophobic motifs. We show that these compounds preferentially label tonoplasts in living Arabidopsis and tobacco (Nicotiana tabacum SR1) cells. ACAIN-labeled membranes co-localized with the GFP signal in plants expressing GFP-δ-TIP (TIP2;1) (a tonoplast aquaporin) fusion protein. ACAIN preserved the dynamics of vacuolar structures. tip2;1 and triple tip1;1-tip1;2-tip2;1 knockout mutants showed weaker ACAIN signal in tonoplasts. The fluorochrome is also suitable for the labeling and detection of specific (cysteine-rich, hydrophobic) proteins from crude cell protein extracts following SDS-PAGE and TIP mutants show altered labeling patterns; however, it appears that ACAIN labels a large variety of tonoplast proteins. ACAIN/CACAIN could be used for the detection of altered vacuolar organization induced by the heptapeptide natural toxin microcystin-LR (MCY-LR), a potent inhibitor of both type 1 and 2A protein phosphatases and a ROS inducer. As revealed both in plants with GFP-TIP2;1 fusions and in wild-type (Columbia) plants labeled with ACAIN/CACAIN, MCY-LR induces the formation of small vesicles, concomitantly with the absence of the large vegetative vacuoles characteristic for differentiated cells. TEM studies of MCY-LR-treated Arabidopsis cells proved the presence of multimembrane vesicles, with characteristics of lytic vacuoles or autophagosomes. Moreover, MCY-LR is a stronger inducer of small vesicle formation than okadaic acid (which inhibits preferentially PP2A) and tautomycin (which inhibits preferentially PP1). ACAIN and CACAIN emerge as useful novel tools to study plant vacuole biogenesis and programmed cell death.

The Antiepileptic Drug Levetiracetam Protects Against Quinolinic Acid-Induced Toxicity in the Rat Striatum.

Levetiracetam (LVT) is a relatively novel antiepileptic drug (AED) known to act through binding with the synaptic vesicular 2A (SV2A) protein, thus modulating the presynaptic neurotransmitter release. The tryptophan metabolite quinolinic acid (QUIN) acts as an excitotoxin when its brain concentrations reach toxic levels under pathological conditions. Since increased neuronal excitability induced by QUIN recruits degenerative events in the brain, and novel AED is also expected to exert neuroprotective effects in their pharmacological profiles, in this work the effect of LVT (54mg/kg, i.p., administered for seven consecutive days) was tested as a pretreatment against the toxicity evoked by the bilateral intrastriatal injection of QUIN (60nmol/μl) to adult rats. QUIN increased the striatal levels of peroxidized lipids and carbonylated proteins as indexes of oxidative damage 24h after its infusion. In addition, in synaptosomal fractions isolated from QUIN-lesioned rats 24h after the toxin infusion, γ-aminobutyric acid (GABA) release was decreased, whereas glutamate (Glu) release was increased. QUIN also decreased motor activity and augmented the rate of cell damage at 7days post-lesion. All these alterations were significantly prevented by pretreatment of rats with LVT. The results of this study show a neuroprotective role and antioxidant action of LVT against the brain damage induced by excitotoxic events.

Complete genome sequence of a novel bromovirus infecting elderberry (Sambucus nigra L.) in the Czech Republic

The genus Bromovirus currently contains six species whose members have relatively narrow host ranges. In the present work, a new bromovirus infecting elderberry (Sambucus nigra L.) is reported. dsRNA was purified and sequenced by next-generation sequencing, and with minimal additional completion by Sanger sequencing, the full tripartite genome was obtained. RNA1 is 3241 nt long and contains ORF1 (1a protein), RNA2 is 2810 nt long and contains ORF2 (2a protein), and RNA3 is 2244 nt long and contains ORF3a (movement protein) and ORF3b (coat protein, CP), separated by an intercistronic poly(A) stretch. Proteins 1a and 2a showed highest sequence identity (69.9% and 69.4%) to the corresponding proteins of melandrium yellow fleck virus. The coat protein showed highest sequence identity (67.9%) to that of brome mosaic virus. The genome shows a typical bromovirus organisation comprising of all the conserved protein domains within the genus. Phylogenetic analysis supports the assignment of this virus as a new member of the genus Bromovirus, for which the name "sambucus virus S" (SVS) is proposed.

Computational identification of microRNAs and their targets in liver cirrhosis.

Previous studies have revealed that the deregulation of circulating miRNAs is associated with liver cirrhosis. The present study aimed to identify reliable candidate biomarkers to improve the early detection of liver cirrhosis. An integrated analysis of expression profiles of microRNAs (miRNAs/miRs) and mRNAs in liver cirrhosis tissues from the GEO database was performed. Next, the regulatory targets of the differentially expressed miRNAs in liver cirrhosis tissues were predicted. In addition, a regulatory network of miRNA-target genes was constructed. A total of 4 eligible mRNA expression profiling studies and 2 miRNA expression profiling studies met the inclusion criteria, and were thus included. A total of 48 differentially expressed miRNAs and 1,773 differentially expressed genes were identified in liver cirrhosis tissues compared with normal tissues. There were 240 miRNA-target pairs whose expression was negatively correlated. In the miRNA-target regulatory network, overexpression of miR-21 and miR-199a-3p was suggested to be closely associated with the progression of liver cirrhosis. In addition, functional enrichment analysis of the target genes indicated that cell cycle was the most significantly enriched pathway, and the dysregulation of leukemia inhibitory factor, cancerous inhibitor of protein phosphatase 2A and retinoblastoma-associated protein 1 clearly suggested their importance in the development of liver cirrhosis. We hypothesized that miR-21 and miR-199a-3p may be promising non-invasive diagnostic biomarkers for the early diagnosis of liver cirrhosis. The miRNA-target regulatory network may provide additional insight into the current data regarding the role of miRNAs in liver cirrhosis.

The Neurospora crassa PP2A Regulatory Subunits RGB1 and B56 Are Required for Proper Growth and Development and Interact with the NDR Kinase COT1.

COT1 is the founding member of the highly conserved nuclear Dbf2-related (NDR) Ser/Thr kinase family and plays a role in the regulation of polar growth and development in Neurospora crassa and other fungi. Changes in COT1 phosphorylation state have been shown to affect hyphal elongation, branching, and conidiation. The function of NDR protein kinases has been shown to be regulated by type 2A protein phosphatases (PP2As). PP2As are heterotrimers comprised of a catalytic and scaffolding protein along with an interchangeable regulatory subunit involved in determining substrate specificity. Inactivation of the N. crassa PP2A regulatory subunits rgb-1 and b56 conferred severe hyphal growth defects. Partial suppression of defects observed in the rgb-1RIP strain (but not in the Δb56 mutant) was observed in cot-1 phosphomimetic mutants, demonstrating that altering COT1 phosphorylation state can bypass, at least in part, the requirement of a functional RGB1 subunit. The functional fusion proteins RGB1::GFP and B56::GFP predominantly localized to hyphal tips and septa, respectively, indicating that their primary activity is in different cellular locations. COT1 protein forms exhibited a hyperphosphorylated gel migration pattern in an rgb-1RIP mutant background, similar to that observed when the fungus was cultured in the presence of the PP2A inhibitor cantharidin. COT1 was hypophosphorylated in a Δb56 mutant background, suggesting that this regulatory subunit may be involved in determining COT1 phosphorylation state, yet in an indirect manner. Reciprocal co-immunoprecipitation analyses, using tagged COT1, PPH1, RGB1, and B56 subunits established that these proteins physically interact. Taken together, our data determine the presence of a functional and physical link between PP2A and COT1 and show that two of the PP2A regulatory subunits interact with the kinase and determine COT1 phosphorylation state.

PPAR\u03b1-dependent Insig2a overexpression inhibits SREBP-1c processing during fasting

Peroxisome-proliferator-activated receptor alpha (PPARα) and sterol regulatory element-binding protein (SREBP) play a role in regulating cellular fatty acid and cholesterol homeostasis via fatty acid oxidation and lipogenesis. The control of SREBP processing is regulated by the insulin induced gene (INSIG)2a protein, which binds SREBP to prevent SREBP translocation to the Golgi apparatus during nutrient starvation in the liver. However, the regulation of SREBP-1c processing by INSIGs during fasting and the regulatory mechanisms of the mouse Insig2a gene expression have not been clearly addressed. In the present study, we found that Insig2a was upregulated by PPARα in mouse livers and primary hepatocytes during fasting, whereas Insig2a mRNA expression was decreased in the livers of refed mice. A PPAR-responsive element between −126 bp and −114 bp in the Insig2a promoter was identified by a transient transfection assay and a chromatin immunoprecipitation assay; its role in regulation by PPARα was characterised using Pparα-null mice. These results suggest that PPARα is a trans-acting factor that enhances Insig2a gene expression, thereby suppressing SREBP-1c processing during fasting.

Antimicrobial activity and synergism of ursolic acid 3-O-α-L-arabinopyranoside with oxacillin against methicillin-resistant Staphylococcus aureus.

The objective of the present study was to investigate the antibacterial activity of a single constituent, ursolic acid 3-O-α-L-arabinopyranoside (URS), isolated from the leaves of Acanthopanax henryi (Oliv.) Harms, alone and in combination with oxacillin (OXA) against methicillin-resistant Staphylococcus aureus (MRSA). A broth microdilution assay was used to determine the minimal inhibitory concentration(MIC). The synergistic effects of URS and OXA were determined using a checkerboard dilution test and time-kill curve assay. The mechanism of action of URS against MRSA was analyzed using a viability assay in the presence of a detergent and an ATPase inhibitor. Morphological changes in the URS-treated MRSA strains were evaluated via transmission electron microscopy(TEM). In addition, the producing penicillin-binding protein2a (PBP2a) protein level was analyzed using western blotting. The MIC value of URS against MRSA was found to be 6.25µg/ml and there was a partial synergistic effect between OXA and URS. The time-kill growth curves were suppressed by OXA combined with URS at a sub-inhibitory level. Compared to the optical density at 600 nm (OD600) value of URS alone (0.09µg/ml), the OD600 values of the suspension in the presence of 0.09µg/ml URS and 0.00001% TritonX-100 or 250µg/ml N,N'-dicyclohexylcarbodiimide reduced by 56.6 and 85.9%, respectively. The TEM images of MRSA indicated damage to the cell wall, broken cell membranes and cell lysis following treatment with URS and OXA. Finally, an inhibitory effect on the expression of PBP2a protein was observed when cells were treated with URS and OXA compared with untreated controls. The present study suggested that URS was significantly active against MRSA infections and revealed the potential of URS as an effective natural antibiotic.

SLPI and AnxA2 expression in neoplasm-free palatine tonsils is associated with smoking habit of individuals

In order to confirm the inverse correlation between secretory leucocyte protease inhibitor (SLPI) expression, and human papillomavirus (HPV) infection previously observed in head and neck squamous-cell carcinoma, the present study retrospectively investigated the association between SLPI and Annexin A2 (AnxA2) expression, and HPV status in non-neoplastic chronic tonsillitis (n=118), and tonsillar hyperplasia (n=96) tissue. We hypothesised that smoking induces the upregulation of SLPI, resulting in reduced binding of HPV to AnxA2, a known modulator of HPV entry into the cell. SLPI and cyclin-dependent kinase inhibitor 2A (p16INK4A) protein expression was measured using immunohistochemistry in 214 specimens; SLPI and AnxA2 gene expression was measured using reverse transcription-quantitative polymerase chain reaction in 213 cases; and DNA was isolated from all the specimens to determine HPV status. The association between the results of the aforementioned analyses and the smoking habits of patients was analysed. The samples were HPV-negative. p16INK4A expression demonstrated moderate and strong staining in 38, and 0 cases, respectively. SLPI expression presented negative, weak and moderate signals in 163, 45, and 6 cases, respectively. A positive correlation was identified between smoking and SLPI (P=0.0001). Gene expression analysis (n=213) revealed that smoking (n=48) resulted in a significant increase in SLPI and AnxA2 expression. A significant positive correlation between AnxA2 and SLPI, indicating a surplus of AnxA2 in relation to SLPI, was exclusively identified in non-smokers. The data demonstrated that smoking results in increased SLPI and AnxA2 expression also in non-neoplastic tonsillar tissue. The observed surplus of AnxA2 in relation to SLPI identified exclusively in the tonsillar tissue of non-smokers indicates a higher possibility of a successful HPV infection of the tonsillar tissue of non-smokers, given the properties of AnxA2 to function as an infection modulator.

Structures and Corresponding Functions of Five Types of Picornaviral 2A Proteins.

Among the few non-structural proteins encoded by the picornaviral genome, the 2A protein is particularly special, irrespective of structure or function. During the evolution of the Picornaviridae family, the 2A protein has been highly non-conserved. We believe that the 2A protein in this family can be classified into at least five distinct types according to previous studies. These five types are (A) chymotrypsin-like 2A, (B) Parechovirus-like 2A, (C) hepatitis-A-virus-like 2A, (D) Aphthovirus-like 2A, and (E) 2A sequence of the genus Cardiovirus. We carried out a phylogenetic analysis and found that there was almost no homology between each type. Subsequently, we aligned the sequences within each type and found that the functional motifs in each type are highly conserved. These different motifs perform different functions. Therefore, in this review, we introduce the structures and functions of these five types of 2As separately. Based on the structures and functions, we provide suggestions to combat picornaviruses. The complexity and diversity of the 2A protein has caused great difficulties in functional and antiviral research. In this review, researchers can find useful information on the 2A protein and thus conduct improved antiviral research.

C1q/tumor necrosis factor-related protein-3 enhances the contractility of cardiomyocyte by increasing calcium sensitivity

C1q/tumor necrosis factor-related protein-3 (CTRP3) is an adipokine that protects against myocardial infarction-induced cardiac dysfunction through its pro-angiogenic, anti-apoptotic, and anti-fibrotic effects. However, whether CTRP3 can directly affect the systolic and diastolic function of cardiomyocytes remains unknown. Adult rat cardiomyocytes were isolated and loaded with Fura-2AM. The contraction and Ca2+ transient data was collected and analyzed by IonOptix system. 1 and 2μg/ml CTRP3 significantly increased the contraction of cardiomyocytes. However, CTRP3 did not alter the diastolic Ca2+ content, systolic Ca2+ content, Ca2+ transient amplitude, and L-type Ca2+ channel current. To reveal whether CTRP3 affects the Ca2+ sensitivity of cardiomyocytes, the typical phase-plane diagrams of sarcomere length vs. Fura-2 ratio was performed. We observed a left-ward shifting of the late relaxation trajectory after CTRP3 perfusion, as quantified by decreased Ca2+ content at 50% sarcomere relaxation, and increased mean gradient (μm/Fura-2 ratio) during 500–600ms (-0.163 vs. −0.279), 500–700ms (-0.159 vs. −0.248), and 500–800ms (-0.148 vs. −0.243). Consistently, the phosphorylation level of cardiac troponin I at Ser23/24 was reduced by CTRP3, which could be eliminated by preincubation of okadaic acid, a type 2A protein phosphatase inhibitor. In summary, CTRP3 increases the contraction of cardiomyocytes by increasing the myofilament Ca2+ sensitivity. CTRP3 might be a potential endogenous Ca2+ sensitizer that modulates the contractility of cardiomyocytes.

Expression and regulation of type 2A protein phosphatases and alpha4 signalling in cardiac health and hypertrophy

Cardiac physiology and hypertrophy are regulated by the phosphorylation status of many proteins, which is partly controlled by a poorly defined type 2A protein phosphatase-alpha4 intracellular signalling axis. Quantitative PCR analysis revealed that mRNA levels of the type 2A catalytic subunits were differentially expressed in H9c2 cardiomyocytes (PP2ACβ > PP2ACα > PP4C > PP6C), NRVM (PP2ACβ > PP2ACα = PP4C = PP6C), and adult rat ventricular myocytes (PP2ACα > PP2ACβ > PP6C > PP4C). Western analysis confirmed that all type 2A catalytic subunits were expressed in H9c2 cardiomyocytes; however, PP4C protein was absent in adult myocytes and only detectable following 26S proteasome inhibition. Short-term knockdown of alpha4 protein expression attenuated expression of all type 2A catalytic subunits. Pressure overload-induced left ventricular (LV) hypertrophy was associated with an increase in both PP2AC and alpha4 protein expression. Although PP6C expression was unchanged, expression of PP6C regulatory subunits (1) Sit4-associated protein 1 (SAP1) and (2) ankyrin repeat domain (ANKRD) 28 and 44 proteins was elevated, whereas SAP2 expression was reduced in hypertrophied LV tissue. Co-immunoprecipitation studies demonstrated that the interaction between alpha4 and PP2AC or PP6C subunits was either unchanged or reduced in hypertrophied LV tissue, respectively. Phosphorylation status of phospholemman (Ser63 and Ser68) was significantly increased by knockdown of PP2ACα, PP2ACβ, or PP4C protein expression. DNA damage assessed by histone H2A.X phosphorylation (γH2A.X) in hypertrophied tissue remained unchanged. However, exposure of cardiomyocytes to H2O2 increased levels of γH2A.X which was unaffected by knockdown of PP6C expression, but was abolished by the short-term knockdown of alpha4 expression. This study illustrates the significance and altered activity of the type 2A protein phosphatase-alpha4 complex in healthy and hypertrophied myocardium.

Constitutively-stressed yeast strains are high-yielding for recombinant Fps1: implications for the translational regulation of an aquaporin

BackgroundWe previously selected four strains of Saccharomyces cerevisiae for their ability to produce the aquaporin Fps1 in sufficient yield for further study. Yields from the yeast strains spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline) that had been transformed with an expression plasmid containing 249 base pairs of 5′ untranslated region (UTR) in addition to the primary FPS1 open reading frame (ORF) were 10–80 times higher than yields from wild-type cells expressing the same plasmid. One of the strains increased recombinant yields of the G protein-coupled receptor adenosine receptor 2a (A2aR) and soluble green fluorescent protein (GFP). The specific molecular mechanisms underpinning a high-yielding Fps1 phenotype remained incompletely described.ResultsPolysome profiling experiments were used to analyze the translational state of spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); all but gcn5Δ were found to exhibit a clear block in translation initiation. Four additional strains with known initiation blocks (rpl31aΔ, rpl22aΔ, ssf1Δ and nop1Δ) also improved the yield of recombinant Fps1 compared to wild-type. Expression of the eukaryotic transcriptional activator GCN4 was increased in spt3Δ, srb5Δ, gcn5Δ and yTHCBMS1 (supplemented with 0.5 μg/mL doxycycline); these four strains also exhibited constitutive phosphorylation of the eukaryotic initiation factor, eIF2α. Both responses are indicative of a constitutively-stressed phenotype. Investigation of the 5′UTR of FPS1 in the expression construct revealed two untranslated ORFs (uORF1 and uORF2) upstream of the primary ORF. Deletion of either uORF1 or uORF1 and uORF2 further improved recombinant yields in our four strains; the highest yields of the uORF deletions were obtained from wild-type cells. Frame-shifting the stop codon of the native uORF (uORF2) so that it extended into the FPS1 ORF did not substantially alter Fps1 yields in spt3Δ or wild-type cells, suggesting that high-yielding strains are able to bypass 5′uORFs in the FPS1 gene via leaky scanning, which is a known stress-response mechanism. Yields of recombinant A2aR, GFP and horseradish peroxidase could be improved in one or more of the yeast strains suggesting that a stressed phenotype may also be important in high-yielding cell factories.ConclusionsRegulation of Fps1 levels in yeast by translational control may be functionally important; the presence of a native uORF (uORF2) may be required to maintain low levels of Fps1 under normal conditions, but higher levels as part of a stress response. Constitutively-stressed yeast strains may be useful high-yielding microbial cell factories for recombinant protein production.