Heat Shock Protein Beta-1 Research Articles

Prognostic value of the TGFß1 rs4803455 single nucleotide polymorphism and its association with prophylactic cranial irradiation in small cell lung cancer.

e21038 Background: Small cell lung cancer (SCLC) is one of the greatest therapeutic challenges of oncology. Potential associations between single-nucleotide polymorphisms (SNP) in Heat shock protein beta- 1 (HSPB1) and Transforming growth factor (TGFß1) and survival have been investigated. Methods: A prospective multicenter study of 94 SCLC patients treated between 2013 and 2016 was conducted. Several variables clinical, tumour-related, therapeutic, and genetic (9 SNPs of TGFß1 gene and 5 of HSPB1 gene) variables were analyzed. Results: The cohort included 77 men and 17 women with a median age of 61 years. Eighty percent presented with limited stage at diagnosis and received thoracic radiation with a median dose of 45 Gy (BID in 42%). Forty-seven percent received concomitant platinum-based chemotherapy and 57% received prophylactic cranial irradiation (PCI). Overall survival (OS) was 34% at 2 years and 16% at 3 years. In multivariate analysis PCI and the TGFß1 SNP rs4803455 showed a statistically significant association with OS and local control. Patients with the CA genotype of the TGFß1 SNP rs4803455 showed worse OS (HR 2.53; IC 1.22-5.21; p = 0.012) and higher local recurrence (HR 2.26; IC 1.01-5.08;p = 0.048). A combined analysis showed that those patients receiving PCI and carrying the rs4803455:CA genotype had a statistically significant lower OS (p < 0.001) and disease-free survival (p < 0.001) than patients receiving PCI and carrying the rs4803455:AA genotype. Conclusions: Genetic analysis showed that the CA genotype of TGFß1 SNP rs4803455 was associated with worse prognosis in SCLC patients and could be considered as a potential biomarker for OS.

Comparative Proteome Analyses of Ureteropelvic Junction Obstruction and Surrounding Ureteral Tissue

Ureteropelvic junction (UPJ) obstruction is a common problem in children, but its etiology remains unclear. In this study, the proteome profiles of the obstructed segment and its surrounding distal and proximal parts were comparatively evaluated. Twelve children younger than 2 years of age with unilateral intrinsic UPJ obstruction were included. The excised operational tissue was divided into three parts immediately after resection: the obstructed part (Obst), the distal normal ureteral part (Dist), and the proximal part of the obstructed segment (Prox). Proteins extracted from the tissue samples were subjected to two-dimensional gel electrophoresis analysis to identify differentially regulated proteins. Spot analysis revealed that four proteins, namely tropomyosin beta and alpha-1 chains, actin and desmin, were upregulated in Obst in comparison to Dist. A similar analysis between Obst and Prox showed that heat shock protein beta-1 and carbonic anhydrase-1 were upregulated in Obst, while tropomyosin alpha 3 chain and ATP synthase beta were upregulated in Prox. The last comparative analysis between Dist and Prox revealed upregulation of annexin-A5 and annexin-A1 in Dist and vimentin, mitochondrial ATP synthase subunit-beta, peroxiredoxin-2, and apolipoprotein-A1 in Prox. Bioinformatics analysis using the STRING server indicated that the differentially regulated proteins, altogether, point to the changes occurring in muscle filament sliding pathway. When regulations occurring in each group were mutually compared, a change in lipase inhibition activity was detected by STRING. This is the first study scrutinizing changes occurring in protein profiles in UPJ.

Mutations in heat shock protein beta-1 (HSPB1) are associated with a range of clinical phenotypes related to different patterns of motor neuron dysfunction: A case series

BackgroundHeat shock protein beta-1 (HSPB1) is a ubiquitously expressed molecular chaperone that is important in protecting cells against cellular injury. Mutations in this protein are known to cause autosomal dominant hereditary distal axonal neuropathies, including Charcot Marie Tooth disease type 2F (CMT2F) and distal hereditary motor neuropathy (dHMN). However, patients with HSPB1 mutations have also been described with upper motor neuron signs.We present five patients with mutations in HSPB1 who presented with a range of clinical phenotypes related to different patterns of motor neuron dysfunction. Three of these mutations have not been previously reported. MethodsPatients were seen at our neuromuscular or amyotrophic lateral sclerosis (ALS) clinics. Gene sequencing was carried out as part of diagnostic investigations. Detailed clinical and electrophysiologic data was collected. ResultsFive patients had variants of HSPB1. Three patients had a hereditary length-dependent sensori-motor axonal neuropathy consistent with Charcot Marie Tooth type 2 (CMT2); two of these patients carried novel mutations in the C-terminal region (p.Glu186* and p.Pro170Thr). One patient had the clinical picture of ALS and a novel missense mutation (p.Arg27Leu) in the N-terminal region. Another patient had the phenotype of hereditary spastic paraparesis (HSP) associated with a missense mutation (p.Gly84Arg) already described in families with CMT or dHMN. ConclusionThis study describes three novel mutations of HSPB1 and describes two patients with upper motor neurone signs associated with HSPB1 mutations.

An Integrative “Omics” Approach, for Identification of Bona Fides PLK1 Associated Biomarker in Esophageal Adenocarcinoma

The rapid expansion of genome-wide profiling techniques offers the opportunity to utilize various types of information collected in the study of human health and disease. Overexpression of Polo like kinase 1 (PLK1) is associated with esophageal adenocarcinoma (OAC), however biological functions and molecular targets of PLK1 in OAC are still unknown. Here we performed integrative analysis of two "omics" data sources to reveal high-level interactions of PLK1 associated with OAC. Initially, quantitative gene expression (RPKM) was measured from transcriptomics data set of four OAC patients. In parallel, alteration in phosphorylation levels was evaluated in the proteomics data set (mass spectrometry) in OAC cell line (PLK1 inhibited). Next, two "omics" data sets were integrated and through comprehensive analysis possible true PLK1 targets that may serve as OAC biomarkers were assembled. Through experimental validation, small ubiquitin-related modifier 1 (SUMO1) and heat shock protein beta-1 (HSPB1) were identified as novel phosphorylation targets of PLK1. Consequently in vivo, in situ and in silico experiments clearly demonstrated the interaction of PLK1 with putative novel targets (SUMO1 and HSPB1). Identification of a PLK1 dependent biosignature in OAC with high confidence in two omics levels proven the robustness and efficacy of our integrative approach.

Novel HDAC6 Inhibitors Increase Tubulin Acetylation and Rescue Axonal Transport of Mitochondria in a Model of Charcot-Marie-Tooth Type 2F.

Disruption of axonal transport causes a number of rare, inherited axonopathies and is heavily implicated in a wide range of more common neurodegenerative disorders, many of them age-related. Acetylation of α-tubulin is one important regulatory mechanism, influencing microtubule stability and motor protein attachment. Of several strategies so far used to enhance axonal transport, increasing microtubule acetylation through inhibition of the deacetylase enzyme histone deacetylase 6 (HDAC6) has been one of the most effective. Several inhibitors have been developed and tested in animal and cellular models, but better drug candidates are still needed. Here we report the development and characterization of two highly potent HDAC6 inhibitors, which show low toxicity, promising pharmacokinetic properties, and enhance microtubule acetylation in the nanomolar range. We demonstrate their capacity to rescue axonal transport of mitochondria in a primary neuronal culture model of the inherited axonopathy Charcot-Marie-Tooth Type 2F, caused by a dominantly acting mutation in heat shock protein beta 1.

Heat shock protein beta 3 (HSPB3) is an unfavorable molecular biomarker in colorectal adenocarcinoma.

Small heat shock proteins (sHSPs) participate in numerous cellular functions including cell signaling, differentiation, and apoptosis. Deregulation of the physiological expression level of sHSPs has been associated with several malignancies. Heat shock protein beta 3 (HSPB3) is the third member of the sHSP family in human and is mainly expressed in skeletal and smooth muscles. In this study, we investigated the potential prognostic significance of HSPB3 expression in colorectal adenocarcinoma, the most frequent type of colorectal cancer. For this purpose, we isolated total RNA from 188 colorectal adenocarcinoma specimens and 68 paired noncancerous ones. After reverse transcription of 2 μg total RNA, we quantified HSPB3 levels by using an in-house-developed real-time quantitative polymerase chain reaction method, based on the SYBR Green chemistry. Comparison of HSPB3 levels among 68 pairs of colorectal tumors and their adjacent noncancerous mucosae uncovered the downregulation of HSPB3 expression in the majority of malignant colorectal tumors. More importantly, high HSPB3 expression is associated with poor relapse-free survival (RFS) and overall survival (OS) of patients with colorectal adenocarcinoma. Multivariable Cox regression analysis revealed that HSPB3 overexpression could serve as an adverse prognostic biomarker in colorectal adenocarcinoma, independent of tumor location, histological grade, and TNM stage. Patients' stratification according to tumor location, histological grade, and TNM stage revealed that high HSPB3 messenger RNA expression retains its unfavorable prognostic potential regarding OS, in particular groups of patients with substantially different prognosis. In conclusion, high HSPB3 expression is associated with poor RFS and OS of patients with colorectal adenocarcioma, independently of clinicopathological prognosticators.

411P - Comparative analysis of protein profiles of prognosis-associated proteins and KIT-related proteins in gastrointestinal stromal tumour

BackgroundGastrointestinal stromal tumor (GIST) is a type of mesenchymal tumor and has KIT mutations in 80–90% of the cases. The development of tyrosine kinase inhibitors have greatly improved patient outcomes. We have previously described association between lower pfetin (KCTD12) expression and poor prognosis in the GIST patients (Ref 1. Suenara Y et al. Clin Cancer Res2008). In this study, to investigate the protein profiles of kit expression, we performed comprehensive protein expression analysis using the gastric tumor cell lines and surgical specimens in GIST. MethodsWe performed KITgene knockdown using siRNA inthe GIST cell line (T1 cell line) and generated the protein expression profiling data by i-TRAQ. Based on this i-TRAQ protein profiling data from the T1 cell line, we referenced our previously published protein expression analysis databases associated with GIST prognosis in the patient samples (Ref 1.) and compared the protein profile of KIT-associated proteins (T1 cell line) and prognosis-associated protein (patient samples). ResultsIn our study, the i-TRAQ profiling method detected expression of approximately 1500–2000 proteins. In KITknockdown cells, 125 proteins were identified to have statistically different expression (p < 0.05). Based on our previously published GIST prognosis protein profile database (using 17 patient tissue samples), 25 proteins were identified to be statistically significant (p < 0.05). Upon comparing the prognosis database with theKIT knockdown database, 6 proteins were identified as common proteins.In particular, the heat shock protein (HSP) 90-beta, which has been previously shown to contribute to the growth of cancer cells, was identified as a critical protein in the biological behavior of GIST. ConclusionsHSP 90-beta is currently considered as a therapeutic target for HSP 90 inhibitors against imatinib-resistant GIST. Our data suggests that the differentiation and cell growth of GIST may be enhanced by the identified proteins, which are in turn induced by KIT. We believe that our established protein profile data will help in improving the understanding of malignant behaviors in GIST. Clinical trial identificationHSP 90-betaplay an important role in the tumor progression of GISTs. Legal entity responsible for the studyJuntendo University. FundingThis study was supported by Grant-in-Aid from the Japan Society for the Promotion of Science (JSPS) KAKENHI (Grant Number #19K22694, #19H03789 and #15KK0353 to Y.S. ; #17K08730 to T.S.; #17K10987to K.K.; #19K16753 to K.A.; #18K15329 to T.O., # 18K16634 to Y.K.) and Leading Advanced Projects for Medical Innovation (LEAP) (Grant Number # JP18am0001009) and the Practical Research for Innovative Cancer Control (Grant Number # JP18ck0106252) from the Japan Agency for Medical Research and Development. DisclosureAll authors have declared no conflicts of interest.

LOXL2 Upregulates Phosphorylation of Ezrin to Promote Cytoskeletal Reorganization and Tumor Cell Invasion.

Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family and its nonsecreted, catalytically dead spliced isoform L2Δ13, enhance cell migration and invasion, stimulate filopodia formation, modulate the expression of cytoskeletal genes, and promote tumor development and metastasis in vivo. We previously showed that LOXL2 reorganizes the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC) cells, however, the underlying molecular mechanisms were not identified. Here, using interactome analysis, we identified ezrin (EZR), fascin (FSCN1), heat shock protein beta-1 (HSPB1), and tropomodulin-3 (TMOD3) as actin-binding proteins that associate with cytoplasmic LOXL2, as well as with its L2Δ13 variant. High levels of LOXL2 and L2Δ13 and their cytoskeletal partners correlated with poor clinical outcome in patients with ESCC. To better understand the significance of these interactions, we focused on the interaction of LOXL2 with ezrin. Phosphorylation of ezrin at T567 was greatly reduced following depletion of LOXL2 and was enhanced following LOXL2/L2Δ13 reexpression. Furthermore, LOXL2 depletion inhibited the ability of ezrin to promote tumor progression. These results suggest that LOXL2-induced ezrin phosphorylation, which also requires PKCα, is critical for LOXL2-induced cytoskeletal reorganization that subsequently promotes tumor cell invasion and metastasis in ESCC. In summary, we have characterized a novel molecular mechanism that mediates, in part, the protumorigenic activity of LOXL2. These findings may enable the future development of therapeutic agents targeting cytoplasmic LOXL2. SIGNIFICANCE: LOXL2 and its spliced isoform L2Δ13 promote cytoskeletal reorganization and invasion of esophageal cancer cells by interacting with cytoplasmic actin-binding proteins such as ezrin.

Identification of novel proteomics markers involved in ovarian endocrinology of riverine buffalo (Bubalus bubalis)

ABSTRACT Long postpartum anestrus is one of the factors responsible for low reproductive efficiency. The present study was carried out to identify probable follicular fluid protein markers of postpartum anestrum buffaloes by proteomics approach. Ovum pick-up technique was used to collect follicular fluids from 23 buffaloes pertaining to postpartum estrus, anestrus and cystic follicular fluid. Pooled follicular fluids samples from each group were further divided into supernatant and cell lysate and subjected to two-dimensional gel electrophoresis. Densitometric analyses revealed the presence of 767 total protein spot and out of which 34 were unique in the studied groups. With the aid of mass spectrometry, the 34 protein spots were identified and among them apolipoproteins (A-I, A-II, A-IV), serum amyloid A-4, adenylate kinase 2, alpha-fetoprotein, heat shock protein beta-1, protein kinase C-binding protein, complement C1q subcomponent, integrin beta-7 and cadherin-23 have been established to play one or other role in ovarian endocrinology. Moreover, using bioinformatics tool, gene ontology and protein–protein interactions among the identified protein were also analyzed. To our knowledge, this is the first report on identification of proteins involved in postpartum anestrus of buffaloes from India which are to be validated further to establish its relationship with postpartum anestrus.

The Urinary Peptidome as a Noninvasive Biomarker Development Strategy for Prenatal Screening of Down's Syndrome.

Prenatal screening for Down's syndrome based on maternal age, ultrasound measures, and maternal serum biomarkers is recommended worldwide, but the false-positive rate and poor diagnostic performance of these screening tests remain problematic. Genetic analysis of cell-free DNA in maternal blood has been developed as a new prenatal screening for Down's syndrome, but it has a number of limitations, including turnaround time and cost. Prenatal screening diagnostic innovation calls for new tests that are noninvasive, accurate, and affordable. We report original observations on potential peptide biomarkers in maternal urine for screening of fetal Down's syndrome. The peptidome of urine samples from 23 pregnant women carrying Down's syndrome fetuses and 30 pregnant women carrying fetuses with normal karyotype was fractionated by weak cation exchange magnetic beads and analyzed by MALDI-TOF mass spectrometry. Levels of six peptides (m/z 1022.1, 1032.1, 1099.5, 1155.9, 1306.6, and 2365.6) were significantly altered between the case and control groups after controlling for maternal and gestational age. A classification model was constructed based on these candidate peptides that could differentiate fetuses with Down's syndrome from controls with a sensitivity of 95.7%, a specificity of 70.0%, and an area under receiver operating characteristic curves of 0.909 (95% confidence interval, 0.835-0.984). Peptide peaks at m/z 1099.5 and 1155.9 were identified as the partial sequences of alpha-1-antitrypsin and heat shock protein beta-1, respectively. These new findings support the new idea that maternal urinary peptidome offers prospects for noninvasive biomarker discovery and development for the prenatal screening of fetal Down's syndrome.

L-Lactate Dehydrogenase B Chain Associated with Milk Protein Content in Dairy Cows.

Simple SummaryThe aim of this study was to explore the genes associated with milk protein content in dairy cows. Seven down-regulated and three up-regulated proteins were found in isolated milk epithelial cells (MECs) from dairy cows with high milk protein level (group High). l-leucine depletion not only decreased the proteins synthesis (p < 0.05), but also decreased l-lactate dehydrogenase B chain (LDH-B) mRNA expression in bovine mammary alveolar cells MAC-T cells (p < 0.05). This study suggested that LDH-B is negatively associated with the milk protein content of dairy cows and has a positive relationship with l-leucine. These findings may provide some theoretical basis to study individual differences in the milk protein synthesis ability of dairy cows.This study aimed to explore genes associated with milk protein content in dairy cows and their relationships with l-leucine. Ten primiparous Holstein cows (93.8 ± 11.56 milking days) fed the same diet were divided into two groups depending on their milk protein contents (group High, 3.34 ± 0.10%; and group Low, 2.86 ± 0.05%). Milk epithelial cells (MECs) were isolated from the collected morning milk and differentially expressed proteins in MECs were explored by two-dimensional gel electrophoresis (2-DE). Then, the mRNA expression of these proteins was detected by real time PCR in MAC-T cells incubated with three different media named positive control (PC), negative control (NC), and l-leucine depletion (NO-leu). Results showed that ten proteins were differentially expressed in MECs from cows in group High. They included seven down-regulated ones (heat shock protein beta-1 (HSPB1), 78 kDa glucose-regulated protein (GRP-78), l-lactate dehydrogenase B chain (LDH-B), malate dehydrogenase, cytoplasmic (MDH1), annexin I (ANXA1), cytokeratin-7 (CK-7), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)), and three up-regulated ones (prohibitin (PHB), beta casein (CSN2), and alpha S1 casein (CSN1S1)). When l-leucine was depleted from the medium, not only proteins content was lowered (p < 0.05), but also the LDH-B mRNA expression was decreased in MAC-T cells (p < 0.05). In conclusion, LDH-B is negatively associated with the milk protein content of dairy cows and has a positive association with l-leucine.

New family with HSPB8-associated autosomal dominant rimmed vacuolar myopathy.

ObjectiveWe clinically and molecularly characterize a new family with autosomal dominant rimmed vacuolar myopathy (RVM) caused by mutations in the HSPB8 gene.MethodsWe performed whole-exome and whole-genome sequencing in the family. Western blot and immunocytochemistry were used to analyze 3 patient fibroblasts, and findings were compared with their age- and sex-matched controls.ResultsAffected patients have distal and proximal myopathy, with muscle biopsy showing rimmed vacuoles, muscle fiber atrophy, and endomysial fibrosis typical of RVM. Muscle MRI showed severe relatively symmetric multifocal fatty degenerative changes of the lower extremities. We identified a duplication of C at position 515 of the HSPB8 gene (c.515dupC) by whole-genome sequencing, which caused a frameshift with a predicted alternate stop codon p.P173SFS*43 in all affected individuals, resulting in an elongated protein product. Western blot and immunocytochemistry studies revealed reduced expression of heat shock protein beta 8 in patient fibroblasts compared with control fibroblasts, in addition to disrupted autophagy pathology.ConclusionsWe report a novel family with autosomal dominant RVM caused by the c.515dupC mutation of the HSPB8 gene, causing a translational frameshift that results in an elongated protein. Understanding the mechanism for the RVM pathology caused by mutated chaperone will permit novel targeted strategies to alter the natural history progression. As next-generation sequencing becomes more available, additional myopathic families will be identified with HSPB8 mutations.

Abstract 4594: Cytoplasmic LOXL2 regulates actin cytoskeletal organization in esophageal squamous cell carcinoma progression

Abstract Background: Lysyl oxidase-like 2 (LOXL2), a copper-dependent enzyme of the lysyl oxidase family, promotes tumor progression and metastasis. We previously revealed that the tumor-promoting role of LOXL2 is mediated by perturbing the architecture of the actin cytoskeleton in esophageal squamous cell carcinoma (ESCC). However, the molecular mechanisms underlying LOXL2-driven ESCC progression remain elusive, and the cytoskeletal proteins that directly interact with LOXL2 remain uncharacterized. Methods: Knockdown LOXL2 in ESCC cells was used by a lentivirus shRNA targeting LOXL2 and analyzed by RNA-sequencing and bioinformatics analysis. Proteomic analysis following by co-immunoprecipitation and immunofluorescence were applied for identification and validation of interacting partners of LOXL2 and its splicing isoform L2Δ13. Cell migration and invasion were measured by wound healing and transwell assays. By risk score calculations, immunohistochemistry of tissue microarrays were adopted to evaluate the prognosis of ESCC patients. Results: We found that knockdown of LOXL2 in ESCC cells suppresses cell migration and invasion, whereas re-expressions of LOXL2 and its non-enzymatic splicing isoform L2Δ13 rescue these cell behaviors. Silencing of LOXL2 inhibits filopodia formation, and induces changes in the expression of cytoskeleton-associated genes. Our interactome analysis identified four actin-binding proteins, i.e. ezrin (EZR), facsin (FSCN1), heat shock protein beta-1 (HSPB1) and tropomodulin-3 (TMOD3), as novel interactors of LOXL2 and L2Δ13. These novel LOXL2/L2Δ13 interaction networks carry important prognostic values in ESCC, as molecular signatures of combinations of LOXL2/L2Δ13 and their cytoskeletal interactors are associated with clinical outcome in ESCC patients. Mechanistically, the ezrin-LOXL2 interaction promotes PKCα-stimulated phosphorylation of ezrin at Thr 567 (T567), an essential residue for ezrin activation, which enhances cell motility and cell invasion in ESCC. Correlating with this, double-high expression of LOXL2 and phosphorylated ezrin-T567 predicts significantly shorter overall survival in ESCC patients. Conclusion: In conclusion, our findings have uncovered a novel molecular mechanism underlying the tumor-promoting role of cytoplasmic LOXL2, which may open new avenues for the therapeutic targeting of LOXL2 in ESCC. (This study is supported by the Natural Science Foundation of China (No. 81472613) and Li Ka Shing Foundation). Citation Format: Xiuhui Zhan, Jiwei Jiao, Haifeng Zhang, Jianzhong He, Runliu Li, Haiying Zou, Zhiyong Wu, Shaohong Wang, Xiue Xu, Jianyi Wu, Liandi Liao, Yinwei Cheng, Kai Zhang, Gera Neufeld, Liyan Xu, Enmin Li. Cytoplasmic LOXL2 regulates actin cytoskeletal organization in esophageal squamous cell carcinoma progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4594.

A Human Three-Dimensional In Vitro Model of Lens Epithelial Cells as a Model to Study Mechanisms of Drug-Induced Posterior Subcapsular Cataracts.

Purpose: Cataract is a pathological opacification of the lens, which is still one of the leading causes of blindness in the world. Several etiologies are described, among them drug-induced cataract, for example, posterior subcapsular cataract (PSC) after steroid treatment. To investigate different mechanisms of drug-induced cataract a human three-dimensional (3D) lens in vitro model was developed, consisting of immortalized human lens epithelial cells. Methods: These cells were cultivated on 96-well, ultralow attachment plates, where they rapidly form spheroids. By gene expression analysis different markers were observed, which are important to maintain lens transparency, such as ephrin type-A receptor 2 (EphA2) or α-smooth muscle actin (α-SMA). Results: The lens epithelial cells form a spheroid within a few days and show stable expression of important lens marker, and size and viability remain stable up to 26 days in culture. The gene expression of the glucocorticoid-treated spheroids revealed a clear shift in the expression of EphA2, α-SMA, αB-crystallin (CRYAB), and heat shock protein beta-1 (HSPB1). Furthermore, the glucocorticoid treatment did not improve cell survival. Conclusions: This study proposes a useful 3D in vitro model, which expresses important lens markers and is capable of demonstrating features found in drug-induced cataracts. As the viability remains stable over long time, this model can also be used for long-term treatment. The main characteristics are the increased expression of α-SMA, CRYAB, and HSPB1 and the decreased expression of EphA2. The present data provide some first evidence on novel mechanisms involved in glucocorticoid-induced cataracts.

Testis-Specific Isoform of Na/K-ATPase (ATP1A4) Interactome in Raft and Non-Raft Membrane Fractions from Capacitated Bovine Sperm.

The plasma membrane of sperm contains highly dynamic lipid microdomains (rafts), which house signaling proteins with a role in regulating capacitation. We reported that ATP1A4, the testis-specific isoform of Na/K-ATPase, interacted with caveolin-1, Src, epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinases 1/2 (ERK1/2) in raft and non-raft domains of the plasma membrane of bovine sperm during capacitation. The objective of the present study was to use a proteomic approach to characterize the ATP1A4 interactome in rafts and non-rafts from capacitated bovine sperm. The non-raft interactome included hexokinase 1, plakophilin 1, desmoglein 1, 14-3-3 protein ζ/δ, cathepsin D and heat shock protein beta1 proteins exclusively, whereas glutathione S-transferase and annexin A2 were unique to raft interactome. However, a disintegrin and metalloprotease 32 (ADAM 32), histone H4, actin, acrosin, serum albumin and plakoglobin were identified in both raft and non-raft fractions of capacitated sperm. Based on gene ontology studies, these differentially interacted proteins were implicated in cell–cell adhesion, signal transduction, fertilization, metabolism, proteolysis and DNA replication, in addition to acting as transport/carrier and cytoskeletal proteins. Overall, we identified proteins not previously reported to interact with ATP1A4; furthermore, we inferred that ATP1A4 may have a role in sperm capacitation.

Ractopamine-induced changes in the proteome of post-mortem beef &lt;i&gt;longissimus lumborum&lt;/i&gt; muscle

Ractopamine is a beta-adrenergic agonist that is approved for use in beef cattle, pigs and turkeys as a repartitioning agent to increase lean muscle deposition and decrease lipogenesis. Although the effects of dietary ractopamine on the proteome profile of post-mortem pork muscles have been examined, its influence on beef muscle proteome has not been studied. Therefore, the objective of this study was to examine the effect of ractopamine on the proteome profile of post-mortem beef longissimus lumborum (LL) muscle. LL muscle samples were obtained from the carcasses of six (n = 6) steers fed ractopamine (RAC; 400 mg ractopamine hydrochloride for 28 days) and six (n = 6) steers fed no ractopamine (CON). The muscle proteome was analysed using two-dimensional gel electrophoresis and tandem mass spectrometry. Five differentially abundant spots were identified, and all the spots were over-abundant in RAC. The identified proteins were involved in muscle structure development (F-actin-capping protein subunit beta-2; PDZ and LIM domain protein-3), chaperone activity (heat shock protein beta-1), oxygen transport (myoglobin), and glycolysis (L-lactate dehydrogenase A chain). These results suggested that dietary ractopamine could influence the abundance of enzymes associated with muscle development and muscle fibre type shift in beef LL muscle. Keywords: growth promotants, meat quality, proteomics

Cardiovascular Disease-Related Serum Proteins in Workers Occupationally Exposed to Polycyclic Aromatic Hydrocarbons.

Chimney sweeps have higher incidence and mortality of cardiovascular disease (CVD), likely related to their exposure to polycyclic aromatic hydrocarbons (PAH). In order to identify underlying mechanisms of PAH-related CVD, we here investigated whether PAH exposure was associated with levels of putative CVD-related proteins in serum among currently working chimney sweeps. We enrolled 116 chimney sweeps and 125 unexposed controls, all nonsmoking male workers from Sweden. We measured monohydroxylated PAH metabolites in urine by liquid chromatography coupled to tandem mass spectrometry and a panel of 85 proteins in serum using proximity extension assay. Linear regression analysis adjusted for age and body mass index showed that 25 proteins were differentially expressed between chimney sweeps and the controls (p < .05, adjusted for false discovery rate). Of the 25 proteins, follistatin (FS), prointerleukin-16 (IL-16), and heat shock protein beta-1 (HSP 27) showed positive associations with the monohydroxylated metabolites of PAH in a dose-response manner (p < .05). Pathway and gene ontology analyses demonstrated that the differentially expressed proteins were mainly involved in inflammatory response and immunological functions, such as leukocyte migration, cell movement of leukocytes, and adhesion of immune cells. In conclusion, we found a number of putative CVD-related proteins differentially expressed, between PAH-exposed and unexposed individuals, and mainly involved in inflammation and immune function. Our data warrant protective measures to reduce PAH exposure and longitudinal investigations of the protein profile in chimney sweeps and other occupational groups exposed to PAH.

HNRNPAB-regulated lncRNA-ELF209 inhibits the malignancy of hepatocellular carcinoma.

Our previous study demonstrated that heterogeneous nuclear ribonucleoprotein AB (HNRNPAB) is a key gene that facilitates metastasis of hepatocellular carcinoma (HCC). However, the molecular mechanisms behind this relationship are not fully understood. In our study, we utilized long-noncoding RNA (lncRNA) microarrays to identify a HNRNPAB-regulated lncRNA named lnc-ELF209. Our findings from chromatin immunoprecipitation assays indicate that HNRNPAB represses lnc-ELF209 transcription by directly binding to its promoter region. We also analyzed clinical samples from HCC patients and cell lines with quantitative real-time polymerase chain reactions, RNA in situ hybridization and immunohistochemistry, and found that there is a negative relationship between HNRNPAB and lnc-ELF209 expression. Up/downregulation assays and rescue assays indicate that lnc-ELF209 inhibits cell migration, invasion and epithelial-mesenchymal transition regulated by HNRNPAB. This suggests a new regulatory mechanism for HNRNPAB-promoted HCC progression. RNA pull-down and LC-MS/MS were used to determine triosephosphate isomerase, heat shock protein 90-beta and vimentin may be involved in the tumor-suppressed function of lnc-ELF209. Furthermore, we found lnc-ELF209 could stabilize TPI protein expression. We also found that lnc-ELF209 overexpression in HCCLM3 cell resulted in a lower rate of lung metastatic, which suggested a less aggressive HCC phenotype. Collectively, these findings offer new insights into the regulatory mechanisms that underlie HNRNPAB cancer-promoting activities and demonstrate that lnc-ELF209 is a HNRNPAB-regulated lncRNA that may play an important role in the inhibition of HCC progression.

Differentially expressed mRNAs, proteins and miRNAs associated to energy metabolism in skeletal muscle of beef cattle identified for low and high residual feed intake

BackgroundFeed efficiency is one of the most important parameters that affect beef production costs. The energy metabolism of skeletal muscle greatly contributes to variations in feed efficiency. However, information regarding differences in proteins involved in the energy metabolism of the skeletal muscle in beef cattle divergently identified for feed efficiency is scarce. In this study, we aimed to investigate energy metabolism of skeletal muscle of Nellore beef cattle, identified for low and high residual feed intake using a proteomics approach. We further assessed the expression of candidate microRNAs as a one of the possible mechanisms controlling the biosynthesis of the proteins involved in energy metabolism that were differentially abundant between high and low residual feed intake animals.ResultsA greater abundance of 14–3-3 protein epsilon (P = 0.01) was observed in skeletal muscle of residual feed intake (RFI) high animals (RFI-High). Conversely, a greater abundance of Heat Shock Protein Beta 1 (P < 0.01) was observed in the skeletal muscle of RFI-Low cattle. A greater mRNA expression of YWHAE, which encodes the 14–3-3 protein epsilon, was also observed in the skeletal muscle of RFI-High animals (P = 0.01). A lower mRNA expression of HSPB1, which encodes the Heat Shock Protein Beta 1, was observed in the skeletal muscle of RFI-High animals (P = 0.01). The miR-665 was identified as a potential regulator of the 14–3-3 protein epsilon, and its expression was greater in RFI-Low animals (P < .001). A greater expression of miR-34a (P = 0.01) and miR-2899 (P < .001) was observed in the skeletal muscle of RFI-High animals, as both miRNAs were identified as potential regulators of HSPB1 expression.ConclusionOur results show that Nellore cattle divergently identified for feed efficiency by RFI present changes in the abundance of proteins involved in energy expenditure in skeletal muscle. Moreover, our data point towards that miR-665, miR34a and miR-2899 are likely involved in controlling both 14-3-3 epsilon and HSPB1 proteins identified as differentially abundant in the skeletal muscle of RFI-High and RFI-Low Nellore cattle.

Runx2 stimulates neoangiogenesis through the Runt domain in melanoma

Runx2 is a transcription factor involved in melanoma cell migration and proliferation. Here, we extended the analysis of Runt domain of Runx2 in melanoma cells to deepen understanding of the underlying mechanisms. By the CRISPR/Cas9 system we generated the Runt KO melanoma cells 3G8. Interestingly, the proteome analysis showed a specific protein signature of 3G8 cells related to apoptosis and migration, and pointed out the involvement of Runt domain in the neoangiogenesis process. Among the proteins implicated in angiogenesis we identified fatty acid synthase, chloride intracellular channel protein-4, heat shock protein beta-1, Rho guanine nucleotide exchange factor 1, D-3-phosphoglycerate dehydrogenase, myosin-1c and caveolin-1. Upon querying the TCGA provisional database for melanoma, the genes related to these proteins were found altered in 51.36% of total patients. In addition, VEGF gene expression was reduced in 3G8 as compared to A375 cells; and HUVEC co-cultured with 3G8 cells expressed lower levels of CD105 and CD31 neoangiogenetic markers. Furthermore, the tube formation assay revealed down-regulation of capillary-like structures in HUVEC co-cultured with 3G8 in comparison to those with A375 cells. These findings provide new insight into Runx2 molecular details which can be crucial to possibly propose it as an oncotarget of melanoma.