K1 Protein Research Articles

A superoxide-driven redox state promotes geroconversion and resistance to senolysis in replication-stress associated senescence

Using S-phase synchronized RPE1-hTERT cells exposed to the DNA damaging agent, methyl methanesulfonate, we show the existence of a redox state associated with replication stress-induced senescence termed senescence-associated redox state (SA-redox state). SA-redox state is characterized by its reactivity with superoxide-sensing fluorescent probes such as dihydroethidine, lucigenin and mitosox and peroxynitrite or hydroxyl radical sensing probe hydroxyphenyl fluorescein (HPF) but not the hydrogen peroxide (H2O2) reactive fluorescent probe CM-H2DCFDA. Measurement of GSH and GSSH also reveals that SA-redox state mitigates the level of total GSH rather than oxidizes GSH to GSSG. Moreover, supporting the role of superoxide (O2.-) in the SA-redox state, we show that incubation of senescent RPE1-hTERT cells with the O2.- scavenger, Tiron, decreases the reactivity of SA-redox state with the oxidants’ reactive probes lucigenin and HPF while the H2O2 antioxidant N-acetyl cysteine has no effect. SA-redox state does not participate in the loss of proliferative capacity, G2/M cell cycle arrest or the increase in SA-β-Gal activity. However, SA-redox state is associated with the activation of NF-κB, dictates the profile of the Senescence Associated Secretory Phenotype, increases TFEB protein level, promotes geroconversion evidenced by increased phosphorylation of S6K and S6 proteins, and influences senescent cells response to senolysis. Furthermore, we provide evidence for crosstalk between SA redox state, p53 and p21. While p53 mitigates the establishment of SA-redox state, p21 is critical for the sustained reinforcement of the SA-redox state involved in geroconversion and resistance to senolysis.

Effects of P70 S6 kinase on the ageing process and reproduction of Daphnia pulex (Branchiopoda, Anomopoda)

Abstract Daphnia pulex is a natural food widely used in aquaculture, and S6K plays an important role in protein translation and ageing. To explore the function of S6K in the ageing process of D. pulex, we cloned and analysed the full-length cDNA of S6K. With the growth of D. pulex, the expression of S6K mRNA first increased, then peaked at 10 days, and then decreased. Western blot analysis showed that the S6K protein expression increased from 1 to 5 days of age, but did not change significantly from 5 to 25 days of age. RNAi knockdown of S6K, reduced the expression of S6K mRNA and S6K protein, and it causes changes in other genes associated with ageing in both sexes. S6K may be involved in reproductive transformation, affecting reproductive capacity, thus significantly affecting the ageing of D. pulex. miRNA with potential regulation of the S6K gene were screened by the bioinformatics method and validated using a dual luciferase reporter gene system. This study deepens our understanding of the mechanisms of ageing in D. pulex and expands our understanding of the role of S6K in the ageing process.

Phosphoproteomics Profiling Defines a Target Landscape of the Basophilic Protein Kinases AKT, S6K, and RSK in Skeletal Myotubes.

Phosphorylation-dependent signal transduction plays an important role in regulating the functions and fate of skeletal muscle cells. Central players in the phospho-signaling network are the protein kinases AKT, S6K, and RSK as part of the PI3K-AKT-mTOR-S6K and RAF-MEK-ERK-RSK pathways. However, despite their functional importance, knowledge about their specific targets is incomplete because these kinases share the same basophilic substrate motif RxRxxp[ST]. To address this, we performed a multifaceted quantitative phosphoproteomics study of skeletal myotubes following kinase inhibition. Our data corroborate a cross talk between AKT and RAF, a negative feedback loop of RSK on ERK, and a putative connection between RSK and PI3K signaling. Altogether, we report a kinase target landscape containing 49 so far unknown target sites. AKT, S6K, and RSK phosphorylate numerous proteins involved in muscle development, integrity, and functions, and signaling converges on factors that are central for the skeletal muscle cytoskeleton. Whereas AKT controls insulin signaling and impinges on GTPase signaling, nuclear signaling is characteristic for RSK. Our data further support a role of RSK in glucose metabolism. Shared targets have functions in RNA maturation, stability, and translation, which suggests that these basophilic kinases establish an intricate signaling network to orchestrate and regulate processes involved in translation.

L-Theanine Regulates the Abundance of Amino Acid Transporters in Mice Duodenum and Jejunum via the mTOR Signaling Pathway

The intestine is a key organ for the absorption of amino acids. L-theanine (LTA) is a structural analog of glutamine and a characteristic non-protein amino acid found in tea (Camellia sinensis) that regulates lipid and protein metabolism. The present study explored the role of LTA in intestinal amino acid absorption, protein synthesis, and its mechanisms. Overall, our findings suggest that LTA supplementation not only affects serum alkaline phosphatase (AKP), total protein (TP), and urea nitrogen (BUN) levels, but it also upregulates the mRNA and protein expression of amino acid transporters (EAAT3, EAAT1, 4F2hc, y+LAT1, CAT1, ASCT2, and B0AT1), and activates the mTOR signaling pathway. The downstream S6 and S6K1 proteins are regulated, and the expression of amino acid transporters is regulated. These findings suggest that LTA increases intestinal AA absorption, promotes protein metabolism, and increases nitrogen utilization by upregulating AAT expression, activating the mTOR signaling pathway, and phosphorylating the mTOR downstream proteins S6 and S6K1.

Association of mTORC1‑dependent circulating protein levels with cataract formation: a mendelian randomization study

BackgroundThe mechanistic target of rapamycin (mTOR) signal pathway plays a critical regulating role in the occurrence and development of cataract. However, the role of mTORC1 downstream proteins, including ribosomal protein S6K (RP-S6K), eukaryotic initiation factor 4E-binding protein (EIF4EBP), eukaryotic initiation factor 4G (EIF-4G), eukaryotic initiation factor 4E (EIF-4E), and eukaryotic initiation factor 4A (EIF-4A), in regulating cataract development is still unknown. Herein, we conducted a mendelian randomization (MR) study to understand the function of mTORC1 signaling in the process of cataract development.ResultsThe causal estimate was evaluated with inverse-variance weighted (IVW) estimate, weighted median estimator, MR-Egger and MR robust adjusted profile score (MR. RAPS). The single-nucleotide polymorphisms (SNPs), P<5 × 10− 6 and r2<0.05, were selected to genetically predict the RP-S6K, EIF4EBP, EIF-4E, EIF-4A, and EIF-4G. We included a total of 26,758 cases and 189,604 controls in this MR study. The study revealed causal association between circulating EIF4EBP (OR 1.09, 95% confidence interval 1.03,1.16, P = 0.004), RP-S6K (OR 1.04, 95% confidence interval 1.01, 1.08, P = 0.02) and cataract formation with IVW estimate. Whereas after correcting outliers, MR robust adjusted profile score (MR. RAPS) shows consistent result with IVW for EIF4EBP (OR = 1.08, 95%CI:1.05–1.11, P = 0.007). The observation strengthened the confidence in the true causal associations. However, no association was found for circulating EIF-4E (OR 1.03, 95% confidence interval 0.97, 1.09, P = 0.31), EIF-4A (OR 1.02, 95% confidence interval 0.98, 1.07, P = 0.34), and EIF-4G (OR 1.02, 95% confidence interval 0.94, 1.01, P = 0.64) levels with cataract formation. No evidence of heterogeneity and unbalanced horizontal pleiotropy was detected.ConclusionThe MR study suggests that EIF4EBP is a high-risk factor for cataract development. There may be a potential causal association between the mTORC1/EIF4EBP axis and cataract. This research highlights the potential mechanism for cataract development and a genetic target to prevent as well as treat cataracts.

Enhancing autophagy and energy metabolism in the meniscus can delay the occurrence of PTOA in ACLT rat.

Osteoarthritis (OA) is a progressive degenerative joint disease characterized by the destruction of the articular cartilage, meniscus and the like. Autophagy and cellular energy metabolism are the mechanisms by which cells maintain homeostasis. However, little is known about the effects of autophagy and cellular energy metabolism on meniscus degeneration, and the pathogenesis of posttraumatic osteoarthritis (PTOA) after the meniscal injury is rarely reported. Therefore, this study aimed to investigate the relationship between changes in autophagy and cellular energy metabolism in the meniscus following anterior cruciate ligament transection (ACLT) and PTOA induced by subsequent articular cartilage injury. In this study, we use a combination of cell experiments in vitro and animal experiments in vivo. On the one hand, cell experiment results show that inhibiting the mTORC1 signaling pathway by inhibiting the phosphorylation of S6K and AKT proteins in meniscal cells will lead to the increase of Beclin1, LC-3B, ATG12, ULK1, P62, and activate autophagy-related signaling pathways, which in turn protects the extracellular matrix component COL1 of meniscal cells from degradation by catabolic factor MMP13. In addition, it increased the generation of mitochondrial membrane potential in meniscal cells, increased the expression of anti-apoptotic factor BCL-XL, decreased the expression of pro-apoptotic factors BAD and BAX, and reduced the apoptosis of meniscal cells. More importantly, under the stimulation of inflammatory factor IL-1β, the secretion of meniscus cells can reduce the elevated levels of MMP13 and Adamts5 caused by chondrocytes affected by IL-1β. On the other hand, the results of animal experiments in vivo further proved the validity of the results of the cell experiments, and also proved that the meniscus injury did prior to the articular cartilage degeneration after ACLT. In conclusion, this study suggests that the meniscus prior to articular cartilage damage during the development of PTOA after ACLT, and that promoting autophagy and energy metabolism of meniscal cells may be a potential therapeutic target for delaying PTOA.

Tight junction disruption through activation of the PI3K/AKT pathways in the skin contributes to blister fluid formation after severe tibial plateau fracture.

Background: Acute compartment syndrome (ACS) is an orthopedic emergency that commonly occurs after severe tibial plateau fracture. Fracture blisters form on the skin, and it was found in our previous study that when blisters form, the compartment pressure significantly decreases. However, the potential mechanism underlying this pressure decrease has not yet been elucidated. Methods: To obtain a comprehensive understanding of the changes that occur after blister formation on the skin, the changes in tight junction expression in the skin after tibial plateau fracture were observed. Blister samples and normal skin were collected from patients with bicondylar tibial plateau fractures with or without blisters. The epidermis thickness was measured, and the difference in the levels of K1, K5, K10, and skin barrier proteins such as claudin 1, claudin 2, and occludin between the two groups was evaluated by immunochemistry analysis, immunofluorescence, Western blotting, and qPCR. Results: The skin was thinner and the levels of K1, K5, and K10 were significantly decreased in blistered skin. Furthermore, the PI3K/AKT pathway was found to be activated, and the tight junction expression was significantly decreased in blistered skin. This indicates that the paracellular pathway, which is essential for accelerating fluid accumulation in blisters and indirectly decreases compartment pressure, was activated. Conclusion: Changes in the tight junction expression after blister formation may underlie blister fluid formation and indirectly explain the decrease in compartment pressure under blistered skin after severe tibial plateau fracture.

Allele-Specific Inactivation of an Autosomal Dominant Epidermolysis Bullosa Simplex Mutation Using CRISPR-Cas9.

Epidermolysis bullosa simplex (EBS) is a rare mechanobullous disease caused by dominant-negative mutations in either keratin 5 (KRT5) or keratin 14 (KRT14) genes. Until now, there is no cure for EBS and the care is primarily palliative. The discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system raised hope for the treatment of EBS and many other autosomal dominant diseases by mutant allele-specific gene disruption. In this study, we aim to disrupt the mutant allele for the heterozygous EBS pathogenic variation c.449T>C (p.Leu150Pro) within KRT5. This mutation generates, naturally, a novel protospacer-adjacent motif for the endonuclease Streptococcus pyogenes Cas9. Thus, we designed a single-guide RNA that guides the Cas9 to introduce a DNA cleavage of the mutant allele in patient's keratinocytes. Then, transfected cells were single-cell cloned and analyzed by deep sequencing. The expression of KRT5 and KRT14 was quantified, and the keratin intermediate filament stability was assessed. Results showed successful stringent mutant allele-specific knockout. An absence of synthesis of mutant transcript was further confirmed indicating permanent mutant allele-specific inactivation. Edited EBS patient keratinocytes produced a lower amount of K5 and K14 proteins compared with nonedited EBS cells, and no disturbance of cellular properties was observed.

478 CRISPER/Cas 9 mediated introduction of the keratin 1 mutation in mice that are identified in patients with epidermolytic ichthyosis

Mutations in the KRT1 cause epidermolytic ichthyosis (EI). There is a strong demand for the development of novel treatment measures. A mutation of c. 1436 T >C; p. Ile 479 Thr of K1 was identified in our case with severe palmoplantar keratoderma, and exaggerated skin lesion on joint skin. Our in silico survey revealed that this amino acid change caused a severe conformational alteration in the K1 protein, strongly suggesting that it interfered with the formation of the K1 / K10 coiled- coil complex.

Effect of polydatin on the proliferation and apoptosis of THP-1 cells and the mechanism

To explore the effect of polydatin on the proliferation and apoptosis of acute monocytic leukemia cell line THP-1 and the possible mechanism. After THP-1 cells were treated with polydatin at gradient concentrations for 24 hours and 48 hours, their proliferation was determined by CCK-8 assay, and half maximal inhibitory concentration (IC50) was calculated. Logarithmically growing THP-1 cells were divided into two groups, a polydatin treatment group (treated with IC50 of polydatin) and a blank control group (treated without polydatin solution), and incubated for 48 hours. Cell apoptosis and cell cycle were measured by flow cytometry. The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins were measured by Western blotting. After treatment with polydatin, the proliferation of THP-1 cells was strongly inhibited, and the IC50 at 48 hours was 1 800 μmol/L. After treatment with 1 800 μmol/L polydatin solution for 48 hours, the apoptosis rate of THP-1 cells increased significantly compared with the blank control group (P<0.05). The cell cycle was arrested in the G0/G1 and S phases, with a significantly increased proportion of cells in the G0/G1 phase and a significantly decreased proportion of cells in the S phase, as compared with the blank control group (P<0.05). The expression levels of PI3K, AKT, p-AKT, mTOR, p-mTOR, p70 S6K, and p-p70 S6K proteins decreased significantly compared with the blank control group (P<0.05). Polydatin can effectively inhibit the proliferation, block the cell cycle, and induce the apoptosis of THP-1 cells, which may be related to inhibition of the PI3K/AKT/mTOR signaling pathway.

Genome-wide regulation of KSHV RNA splicing by viral RNA-binding protein ORF57.

RNA splicing plays an essential role in the expression of eukaryotic genes. We previously showed that KSHV ORF57 is a viral splicing factor promoting viral lytic gene expression. In this report, we compared the splicing profile of viral RNAs in BCBL-1 cells carrying a wild-type (WT) versus the cells containing an ORF57 knock-out (57KO) KSHV genome during viral lytic infection. Our analyses of viral RNA splice junctions from RNA-seq identified 269 RNA splicing events in the WT and 255 in the 57KO genome, including the splicing events spanning large parts of the viral genome and the production of vIRF4 circRNAs. No circRNA was detectable from the PAN region. We found that the 57KO alters the RNA splicing efficiency of targeted viral RNAs. Two most susceptible RNAs to ORF57 splicing regulation are the K15 RNA with eight exons and seven introns and the bicistronic RNA encoding both viral thymidylate synthase (ORF70) and membrane-associated E3-ubiquitin ligase (K3). ORF57 inhibits splicing of both K15 introns 1 and 2. ORF70/K3 RNA bears two introns, of which the first intron is within the ORF70 coding region as an alternative intron and the second intron in the intergenic region between the ORF70 and K3 as a constitutive intron. In the WT cells expressing ORF57, most ORF70/K3 transcripts retain the first intron to maintain an intact ORF70 coding region. In contrast, in the 57KO cells, the first intron is substantially spliced out. Using a minigene comprising of ORF70/K3 locus, we further confirmed ORF57 regulation of ORF70/K3 RNA splicing, independently of other viral factors. By monitoring protein expression, we showed that ORF57-mediated retention of the first intron leads to the expression of full-length ORF70 protein. The absence of ORF57 promotes the first intron splicing and expression of K3 protein. Altogether, we conclude that ORF57 regulates alternative splicing of ORF70/K3 bicistronic RNA to control K3-mediated immune evasion and ORF70 participation of viral DNA replication in viral lytic infection.

Abstract 4: Intercepting ribosomal protein S6KB1 signaling: Prevention of prostate cancer recurrence

Abstract There is an urgent need for innovative strategies such as the discovery of adjuvants that can prevent relapse and improve quality of life for patients treated with radiotherapy. Previously we demonstrated the utility of Nexrutine (Nex) as a neo-adjuvant with radiation. Nex was safe and well tolerated in PCa patients and potentiated radiation response in part through downregulation of ribosomal protein S6K (encoded byRPS6KB1). We now show that RPS6KB1 depleted prostate cancer cells with higher basal levels of γ-H2AX, a marker for DNA double strand breaks (i) are more sensitive to radiation and (ii) form smaller tumors with reduced levels of prostate specific antigen (PSA). Depletion of RPS6KB1 hindered DNA double-strand break repair predominantly through the alternate end-joining pathway, induction of G2/M checkpoint and NFκB pathway activation. Collectively these events led to improved radiation sensitivity. We further identified Berberine (Ber), one of the active constituents of Nex as a potential pharmacological inhibitor of RPS6KB1. In an orthotopic implantation model of C4-2B, treatment with Ber alone or Ber plus radiation decreased PSA levels that was sustained during the course of the experiment. On the other hand animals treated with radiation alone developed recurrent cancer as evidenced by a resurgence of PSA. Animals administered Ber followed by XRT intervention had increased levels of RANTES while there was no change in animals that received XRT followed by Ber. The observed reversal of the Bereffect with the sequence of intervention is statistically significant (p=0.0298). Among animals not subject to XRT, the mean PSA increased in those that did not receive Ber relative to those that did; mean difference=-1.93, 95% CI -3.75 to -0.105, p=0.04 with no significant changes in body weight. Notably,RPS6KB1 mRNA levels increased in tumor samples in patients experiencing biochemical recurrence(BCR). Given that rising PSA following conventional therapeutic approaches such as radiation remain a major clinical challenge, targeting RPS6KB1 signaling with radiation therapy is an attractive strategy to prevent BCR. Supported in part by CPRIT RP190012 (APK). Citation Format: Addanki Pratap Kumar, Alison Clark, Michelle Villarreal, Sridharan Jayamohan, Shih-Bo Huang, Suleman S. Hussain, Xiaoyu Yang, Paul Rivas, Darpan Patel, Bethany L. Pierce, Shreya Tripathy, Pawel Osmulski, Maria Gaczynska, Lai Zhao, Li-Ju Wang, Yidong Chen, Caroline Xavier Paul Ezhilan, Mohan Natarajan, Joel E. Michalek, Robert L. Reddick, Rita Ghosh. Intercepting ribosomal protein S6KB1 signaling: Prevention of prostate cancer recurrence [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 4.

Blocking autophagy with chloroquine aggravates lipid accumulation and reduces intracellular energy synthesis in hepatocellular carcinoma cells, both contributing to its anti-proliferative effect

PurposeThe autophagy inhibitor chloroquine enhances the effect of targeted therapy using tyrosine kinase inhibitor in liver cancer. We would like to further understand the specific mechanism by which chloroquine inhibits the proliferation of tumor cells.MethodsWe used a human hepatocarcinoma cell line (HepG2) as cell culture model. In contrast to the control groups (treated only with complete medium), cells in experimental groups were treated either with complete medium + 40 ng/ml Hepatocyte growth factor (HGF), or with complete medium + 60 μM chloroquine or with complete medium + 40 ng/ml HGF + 60 μM chloroquine for 24 h. Cell number and ATP content were investigated using spectrophotometric assays. Cell proliferation and apoptosis were detected by immunohistochemistry. Cell morphological alterations were examined by Giemsa and H&E staining. Cellular lipid content was determined by Oil Red O staining and Triglyceride quantification assay. Autophagy-related proteins (LC3B and p62) and hepatocyte proliferation-related protein (S6K1) were examined using western blot. The autophagic flux of cells was assessed by mRFP-EGFP-LC3 transfection assay.ResultsWe found that chloroquine inhibited the proliferation of HepG2 cells, as evidenced by a decrease in cellular ATP content, Ki-67 and S6K1 protein expression and a reduction in cell number. This finding was associated with an increase in lipid content. As expected, chloroquine inhibited autophagy of HepG2 cells, as evidenced by the accumulation of LC3B-II and the significant upregulation of p62. mRFP-EGFP-LC3 transfection assay showed that indeed chloroquine blocked the autophagic flux in HepG2 cells.ConclusionChloroquine impaired proliferation of HepG2 cells might be due to intracellular accumulation of lipids and inhibition of energy synthesis.

Stress keratin 17 as a novel biomarker of response in immune checkpoint blockade–treated head and neck squamous cell carcinoma.

3117 Background: Low response rates in immune checkpoint blockade (ICB) treated head and neck squamous cell carcinoma (HNSCC)drive a critical need for robust clinically validated biomarkers that can predict response to ICB. Stress keratin 17 (K17) is a known prognostic marker in various types of cancer, including HNSCC; however, its predictive value for ICB response has not been investigated. Preclinical studies suggest K17 suppresses macrophage-mediated CXCL9/CXCL10 chemokine signaling involved in attracting activated CD8+ T cells into tumors. Furthermore, knocking out K17 results in restored response to ICB in a HNSCC mouse model. Here, we evaluated if K17 protein expression predicts response to ICB in human HNSCC patients. Methods: We conducted a retrospective analysis of 26 HNSCC patients that received at least one cycle of pembrolizumab at the University of Wisconsin-Madison Carbone Cancer Center. Pretreatment, archival, formalin-fixed, paraffin-embeded samples were stained by immunohistochemistry using a K17 monoclonal antibody. Clinical outcomes were investigator-assessed for all patients with at least one post-treatment scan or evidence of clinical progression after treatment initiation. Based on independent pathology review, cases were categorized into K17 high vs K17 low based on a cut-off of &gt; 5% strong cytoplasmic staining intensity of tumor cells in the invasive carcinoma component. Correlation between K17 expression and clinical outcomes was assesed using Fischer's exact test and log rank test. Results: The 26 patients included in this study were 85% male, median age 60.5 years, 74% ECOG performance status &lt; 2, with 80% having received prior chemotherapy. Primary site included oral cavity (54%), oropharynx (23%), larynx (4%), or other (19%). Seventeen tumors (65%) showed high K17 expression, and 9 tumors (35%) showed low K17 expression. Eleven patients (42%) had programmed death ligand 1 (PD-L1)+ tumors as determined by combined positive score. Six patients (23%, all K17 low) achieved clinical benefit, while 20 patients (77%, 17 K17 high and 2 K17 low) had progressive disease. High K17 expression was associated with lack of clinical benefit (p &lt; 0.001), shorter time to treatment failure (p &lt; 0.001), progression-free (p = 0.004) and overall survival (p = 0.02). PD-L1 expression by immunohistochemistry (clone 22C3) did not correlate with K17 expression or clinical outcome. Conclusions: Our findings suggest that K17 expression may predict clinical benefit from ICB in HNSCC patients, thus supporting further validation studies. [Table: see text]

Effect of Cyclic Heat Stress on Hypothalamic Oxygen Homeostasis and Inflammatory State in the Jungle Fowl and Three Broiler-Based Research Lines.

Heat stress (HS) is devastating to poultry production sustainability due its detrimental effects on performance, welfare, meat quality, and profitability. One of the most known negative effects of HS is feed intake depression, which is more pronounced in modern high-performing broilers compared to their ancestor unselected birds, yet the underlying molecular mechanisms are not fully defined. The present study aimed, therefore, to determine the hypothalamic expression of a newly involved pathway, hypoxia/oxygen homeostasis, in heat-stressed broiler-based research lines and jungle fowl. Three populations of broilers (slow growing ACRB developed in 1956, moderate growing 95RB from broilers available in 1995, and modern fast growing MRB from 2015) and unselected Jungle fowl birds were exposed to cyclic heat stress (36°C, 9 h/day for 4 weeks) in a 2 × 4 factorial experimental design. Total RNAs and proteins were extracted from the hypothalamic tissues and the expression of target genes and proteins was determined by real-time quantitative PCR and Western blot, respectively. It has been previously shown that HS increased core body temperature and decreased feed intake in 95RB and MRB, but not in ACRB or JF. HS exposure did not affect the hypothalamic expression of HIF complex, however there was a line effect for HIF-1α (P = 0.02) with higher expression in JF under heat stress. HS significantly up regulated the hypothalamic expression of hemoglobin subunits (HBA1, HBBR, HBE, HBZ), and HJV in ACRB, HBA1 and HJV in 95RB and MRB, and HJV in JF, but it down regulated FPN1 in JF. Additionally, HS altered the hypothalamic expression of oxygen homeostasis- up and down-stream signaling cascades. Phospho-AMPKThr172 was activated by HS in JF hypothalamus, but it decreased in that of the broiler-based research lines. Under thermoneutral conditions, p-AMPKThr172 was higher in broiler-based research lines compared to JF. Ribosomal protein S6K1, however, was significantly upregulated in 95RB and MRB under both environmental conditions. HS significantly upregulated the hypothalamic expression of NF-κB2 in MRB, RelB, and TNFα in ACRB, abut it down regulated RelA in 95RB. The regulation of HSPs by HS seems to be family- and line-dependent. HS upregulated the hypothalamic expression of HSP60 in ACRB and 95RB, down regulated HSP90 in JF only, and decreased HSP70 in all studied lines. Taken together, this is the first report showing that HS modulated the hypothalamic expression of hypoxia- and oxygen homeostasis-associated genes as well as their up- and down-stream mediators in chickens, and suggests that hypoxia, thermotolerance, and feed intake are interconnected, which merit further in-depth investigations.

Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE

The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family, governs various cellular processes by phosphorylating a large set of substrates. Although many studies have expanded the number of ERK substrates, it is likely that additional substrates remain to be discovered. Here we have employed a quantitative phosphoproteomic approach to explore novel ERK substrates in NIH3T3 fibroblasts stably expressing a fusion protein between B-Raf and estrogen receptor. Among ERK-dependent phosphorylation targets, we focused on NGFI-A-binding protein 2 (Nab2), forkhead box protein K1 (Foxk1), and Disks large-associated protein 5 (Dlgap5/HURP). Phos-tag SDS-PAGE followed by Western blotting confirmed ERK-dependent phosphorylation of these three proteins in cells. Phos-tag SDS-PAGE of in vitro kinase assay samples revealed high degrees of phosphorylation of these proteins by active ERK. Furthermore, in-gel digestion of the phosphorylated protein bands from Phos-tag SDS-PAGE followed by LC-MS/MS indicated that active ERK directly phosphorylates the same sites in vitro as those observed in cells. This study demonstrates the usefulness of Phos-tag SDS-PAGE for validation of candidate substrates of protein kinases. SignificanceLabel-free quantitative phosphoproteomics identified 1439 phosphopeptides derived from 840 proteins that were significantly increased by ERK activation in mouse fibroblasts. Through gene ontology and pathway analysis, we selected three proteins involved in transcriptional regulation and/or tumorigenesis. The identified phosphorylation sites of these proteins conform to the ERK consensus motif and were directly phosphorylated by active ERK in vitro. Phos-tag SDS-PAGE was useful for detecting ERK-mediated phosphorylation of these substrates both in cells and in vitro. Further characterization of these new ERK substrates will be needed to better understand the ERK signaling pathway, and our phosphoproteomic data provide useful information for studying downstream substrates of ERK.

Homogalacturonan from squash: Characterization and tau-binding pattern of a sulfated derivative

A pectic polysaccharide (WAP) was isolated from squash and identified as a homogalacturonan with a molecular mass of 83.2 kDa by GPC, monosaccharide composition analysis, FT-IR and NMR spectra. Sulfation modification of WAP was carried out and a sulfated derivative (SWAP) was obtained with a substitution degree of 1.81. The NMR spectrum indicated that the sulfation modification mainly occurred at the C-2 and C-3 positions of galacturonan residues. The binding pattern of SWAP to tau K18 protein was observed in 2D 1H15N HSQC spectra of tau, which resembled the tau-heparin interaction, with R2 domain as the major binding region. These results suggest that SWAP has the potential to act as a heparin mimic to inhibit the transcellular spread of tau; thus natural polysaccharide from squash may be developed into therapies for AD and related tauopathies.

Intersectins as Novel Protein for Viral Endocytosis- A Mini Review

Intersectins (INSTNs) have been defined as scaffold proteins that have several biochemical functions. Initially INSTNs are described as endocytic regulators and function in different signalling pathways including Ras and Ras-like GTPases, PI3Ks and RTKs in several diseases. Recently, INSTNs have been reported in viral endocytosis. INSTNs was found to be upregulated in transcriptomic analysis of differentially expressed protein from Dengue virus serotype four from our recent data. Hence, this review summarises the role of INSTNs in viral endocytosis by interaction with signalling pathways. INSTN-2 interact with K15 protein of Kaposi’s Sarcoma-Associated Herpesvirus by selective SH3 domain, INSTN-1 interaction with Epstein-Barr virus Latent Membrane Protein 2 (LMP2A), while direct interaction of NPF motifs at C-terminus of A36 with INSTN-1 and Epsin15 homology domains recruiting AP-2 and clathrin mediate viral release was reported in Vaccinia virus. Most recent, INSTN-2 was shown to regulate adaptive immune response during viral infectivity. This was discovered when mice deficient in INSTN-2 exhibited high mortality rate, impaired antibody responses to vaccination and reduced germinal centre formation in Influenza A virus. This review revealed INSTNs as essential viral endocytic proteins. The review also highlights INSTNs role in other diseases and the regulation of signalling pathways. Targeting INSTNs could be important for the development of pharmaceuticals against viruses and diseases. More study is required to establish the mechanistic role of INSTNs for viral endocytosis.

Proteasome-mediated degradation of keratins 7, 8, 17 and 18 by mutant KLHL24 in a foetal keratinocyte model: Novel insight in congenital skin defects and fragility of epidermolysis bullosa simplex with cardiomyopathy

Epidermolysis bullosa simplex (EBS) with cardiomyopathy (EBS-KLHL24) is an EBS subtype caused by dominantly inherited, gain-of-function mutations in the gene encoding for the ubiquitin-ligase KLHL24, which addresses specific proteins to proteasomal degradation. EBS-KLHL24 patients are born with extensive denuded skin areas and skin fragility. Whilst skin fragility rapidly ameliorates, atrophy and scarring develop over time, accompanied by life-threatening cardiomyopathy. To date, pathogenetic mechanisms underlying such a unique disease phenotype are not fully characterized. The basal keratin 14 (K14) has been indicated as a KLHL24 substrate in keratinocytes. However, EBS-KLHL24 pathobiology cannot be determined by the mutation-enhanced disruption of K14 alone, as K14 is similarly expressed in foetal and postnatal epidermis and its protein levels are preserved both in vivo and in vitro disease models. In this study, we focused on foetal keratins as additional KLHL24 substrates. We showed that K7, K8, K17 and K18 protein levels are markedly reduced via proteasome degradation in normal foetal keratinocytes transduced with the mutant KLHL24 protein (ΔN28-KLHL24) as compared to control cells expressing the wild-type form. In addition, heat stress led to keratin network defects and decreased resilience in ΔN28-KLHL24 cells. The KLHL24-mediated degradation of foetal keratins could contribute to congenital skin defects in EBS-KLHL24. Furthermore, we observed that primary keratinocytes from EBS-KLHL24 patients undergo accelerated clonal conversion with reduced colony forming efficiency (CFE) and early replicative senescence. Finally, our findings pointed out a reduced CFE in ΔN28-KLHL24-transduced foetal keratinocytes as compared to controls, suggesting that mutant KLHL24 contributes to patients’ keratinocyte clonogenicity impairment.

O-158 The effect of circadian rhythm disruption due to constant light on ovarian and oocyte aging through possible relationship between PER2 and mTOR signaling pathway

Abstract Study question Does circadian rhythm disruption by constant light affect the ovarian morphology and function, and cause ovarian and oocyte aging through possible relationship between PER2 and mTOR? Summary answer We demonstrated that circadian rhythm disruption by light may cause ovarian and oocyte aging. What is known already Circadian rhythm regulates multiple physiological processes and PER2 is one of the core circadian rhythm components. Changes in light conditions may cause circadian rhythm disruptions. Light exposure at night may cause attenuation in PER2 mRNA and protein levels. Circadian rhythm disruptions are thought to be associated with reproductive diseases. mTOR signaling pathway functions in folliculogenesis and oocyte maturation in ovary. Also, it is associated with ovarian and oocyte aging. Study design, size, duration A total of 32 female Balb/c mice which enter estrous cycle were used in the study. Mice were randomly assigned to one of two groups as 12:12h L:D and 12:12h L:L. During the experiment, 12:12h L:D (control group) was housed in a 12:12h light:dark cycle and 12:12h L:L (experiment group) was housed in a constant light conditions 12:12h light:light for 1 week. Participants/materials, setting, methods We housed 12:12h L:D group in standard lightening conditions and 12:12h L:L group in constant light for one week. We performed food intake and body weight change analysis. We evaluated ovarian morphology, follicle counting analysis. We evaluated ZP3 and nitrotyrosine (NTY) expression for oocyte aging markers. We performed western blot for PER2, mTOR, p-mTOR, p70 S6K, p-p70 S6K, and Caspase-3 protein levels. Main results and the role of chance We demonstrated that circadian rhythm disruption caused alteration in their food intake and decrease in primordial follicle numbers and increase in atretic follicles (p &amp;lt; 0.05). It caused increase in oxidative stress and decrease in ZP3 expression in oocytes (p &amp;lt; 0.05). We showed decreased protein levels of PER2, mTOR, p-mTOR and p70 S6K (p &amp;lt; 0.05). Limitations, reasons for caution The explanation of molecular mechanism underlying the relationship between circadian rhythm disruptions by light and ovarian function may lead the usage of circadian rhythm-based or light-based therapies currently using to treat some diseases on female reproductive system related diseases. Wider implications of the findings We conclude that constant light may reduce follicle reserve, cause follicles to go rapidly atresia and disrupt the oocyte quality, thus it may be a risk factor for female reproductive diseases such as premature ovarian insufficiency and early menopause. Trial registration number not applicable