Protein Annexin Research Articles

Analysis of the acute neuroinflammatory response and endogenous markers in the amygdala of animals submitted to status epilepticus by intrahippocampal pilocarpine application

Introduction: Experimental evidence and clinical evidence indicate that the inflammatory process is a crucial mechanism in the pathophysiology of epileptic seizures and temporal lobe epilepsy. The amygdala when involved in an atypical processing is associated with multiple moods such as depression and anxiety disorder and psychiatric disorders such as schizophrenia. Objective: This study investigated the acute inflammatory process and modulation of the endogenous proteins’ galectins and AnxA1 in the amygdala of animals submitted to an experimental model of temporal lobe epilepsy. Methods: The experimental procedures were approved by the Ethics Committee on the Use of Animals at UNIFESP (CEUA nº2958050814). The experiments performed in this study used data and materials that were obtained from the project “Neuroprotective and anti-inflammatory role of the mimetic peptide ac2-26 of the annexin a1 protein in intrahippocampal pilocarpine-induced status epilepticus” conducted by the advisor. The experimental model used male Wistar rats that were divided into 3 experimental groups (NAIVE; SHAM, Status Epilepticus or SE - n = 5 animals/group). Once acclimated, the animals in the SHAM and SE groups underwent stereotaxic surgery for implantation of the intracerebral cannula in the right hippocampus. The SHAM animals received sterile saline in all procedures and the NAIVE group only manipulated. The animals were monitored throughout the period and after 24 hours of experiment all animals were euthanized by overdose of thiopental to remove the brain and performed histological and immunohistochemical analysis. Results and Conclusion: Initial results demonstrate that SE and the acute inflammatory process cause damage to the amygdala, and there is also modulation of inflammatory markers in this structure. However, further studies are needed to better understand the mechanism of action in neuroinflammation in status epilepticus.

From NSAIDs to Glucocorticoids and Beyond.

Our interest in inflammation and its treatment stems from ancient times. Hippocrates used willow bark to treat inflammation, and many centuries later, salicylic acid and its derivative aspirin’s ability to inhibit cyclooxygenase enzymes was discovered. Glucocorticoids (GC) ushered in a new era of treatment for both chronic and acute inflammatory disease, but their potentially dangerous side effects led the pharmaceutical industry to seek other, safer, synthetic GC drugs. The discovery of the GC-inducible endogenous anti-inflammatory protein annexin A1 (AnxA1) and other endogenous proresolving mediators has opened a new era of anti-inflammatory therapy. This review aims to recapitulate the last four decades of research on NSAIDs, GCs, and AnxA1 and their anti-inflammatory effects.

Quantitative proteomic screen identifies annexin A2 as a host target for Salmonella pathogenicity island-2 effectors SopD2 and PipB2

Intracellular pathogens need to establish an intracellular replicative niche to promote survival and replication within the hostile environment inside the host cell. Salmonella enterica serovar Typhimurium (S. Typhimurium) initiates formation of the unique Salmonella-containing vacuole and an extensive network of Salmonella-induced tubules in order to survive and thrive within host cells. At least six effectors secreted by the type III secretion system encoded within Salmonella pathogenicity island-2 (SPI-2), namely SifA, SopD2, PipB2, SteA, SseJ, and SseF, purportedly manipulate host cell intracellular trafficking and establish the intracellular replicative niche for S. Typhimurium. The phenotypes of these effectors are both subtle and complex, complicating elucidation of the mechanism underpinning host cell manipulation by S. Typhimurium. In this work we used stable isotope labeling of amino acids in cell culture (SILAC) and a S. Typhimurium mutant that secretes increased amounts of effectors to identify cognate effector binding partners during infection. Using this method, we identified the host protein annexin A2 (AnxA2) as a binding partner for both SopD2 and PipB2 and were able to confirm its binding to SopD2 and PipB2 by reciprocal pull down, although there was a low level of non-specific binding of SopD2-2HA and PipB2-2HA to the Ni-Sepharose beads present. We further showed that knockdown of AnxA2 altered the intracellular positioning of the Salmonella containing vacuole (SCV). This suggests that AnxA2 plays a role in the subcellular positioning of the SCV which could potentially be mediated through protein–protein interactions with either SopD2 or PipB2. This demonstrates the value of studying effector interactions using proteomic techniques and natural effector delivery during infection rather than transfection.

The Ca2+- and phospholipid-binding protein Annexin A2 is able to increase and decrease plasma membrane order

Annexin A2 (AnxA2) is a calcium- and phospholipid-binding protein that plays roles in cellular processes involving membrane and cytoskeleton dynamics and is able to associate to several partner proteins. However, the principal molecular partners of AnxA2 are negatively charged phospholipids such as phosphatidylserine and phosphatidyl-inositol-(4,5)-phosphate. Herein we have studied different aspects of membrane lipid rearrangements induced by AnxA2 membrane binding. X-ray diffraction data revealed that AnxA2 has the property to stabilize lamellar structures and to block the formation of highly curved lipid phases (inverted hexagonal phase, HII). By using pyrene-labelled cholesterol and the environmental probe di-4-ANEPPDHQ, we observed that in model membranes, AnxA2 is able to modify both, cholesterol distribution and lipid compaction. In epithelial cells, we observed that AnxA2 localizes to membranes of different lipid order. The protein binding to membranes resulted in both, increases and/or decreases in membrane order depending on the cellular membrane regions. Overall, AnxA2 showed the capacity to modulate plasma membrane properties by inducing lipid redistribution that may lead to an increase in order or disorder of the membranes.

OsANN4 modulates ROS production and mediates Ca2+ influx in response to ABA

BackgroundPlant annexins are calcium- and lipid-binding proteins that have multiple functions, and a significant amount of research on plant annexins has been reported in recent years. However, the functions of annexins in diverse biological processes in rice are largely unclear.ResultsHerein, we report that OsANN4, a calcium-binding rice annexin protein, was induced by abscisic acid (ABA). Under ABA treatment, the plants in which OsANN4 was knocked down by RNA interference showed some visible phenotypic changes compared to the wild type, such as a lower rooting rate and shorter shoot and root lengths. Moreover, the superoxide dismutase (SOD) and catalase (CAT) activities of the RNAi lines were significantly lower and further resulted in higher accumulation of O2.- and H2O2 than those of the wild-type. A Non-invasive Micro-test Technology (NMT) assay showed that ABA-induced net Ca2+ influx was inhibited in OsANN4 knockdown plants. Interestingly, the phenotypic differences caused by ABA were eliminated in the presence of LaCl3 (Ca2+ channel inhibitor). Apart from this, we demonstrated that OsCDPK24 interacted with and phosphorylated OsANN4. When the phosphorylated serine residue of OsANN4 was substituted by alanine, the interaction between OsANN4 and OsCDPK24 was still observed, however, both the conformation of OsANN4 and its binding activity with Ca2+ might be changed.ConclusionsOsANN4 plays a crucial role in the ABA response, partially by modulating ROS production, mediating Ca2+ influx or interacting with OsCDPK24.

Analysis of the myeloid-derived suppressor cells and annexin A1 in multibacillary leprosy and reactional episodes

BackgroundLeprosy is a chronic infectious disease caused by Mycobacterium leprae. Patients have distinct clinical forms, and the host´s immunological response regulate those manifestations. In this work, the presence of the myeloid-derived suppressor cell and the regulatory protein annexin A1 is described in patients with multibacillary leprosy and with type 1 and 2 reactions.MethodsPatients were submitted to skin biopsy for histopathological analysis to obtain a bacilloscopic index. Immunofluorescence was used to detect myeloid-derived suppressor cells and annexin A1.ResultsThe data demonstrated that the presence of granulocytic and monocytic myeloid-derived suppressor cells in leprosy patients. A high number of monocytic myeloid-derived suppressor cells were observed in lepromatous leprosy and type 2 reactional patients. The presence of annexin A1 was observed in all myeloid-derived suppressor cells. In particular, the monocytic myeloid-derived suppressor cell in the lepromatous patients has higher levels of this protein when compared to the reactional patients. This data suggest that the higher expression of this protein may be related to regulatory response against a severe infection, contributing to anergic response. In type 1 reactional patients, the expression of annexin A1 was reduced.ConclusionsMyeloid-derived suppressor cell are present in leprosy patients and annexin A1 might be regulated the host response against Mycobacterium leprae.

Histone modification of pain-related gene expression in spinal cord neurons under a persistent postsurgical pain-like state by electrocautery

Chronic postsurgical pain (CPSP) is a serious problem. We developed a mouse model of CPSP induced by electrocautery and examined the mechanism of CPSP. In this mouse model, while both incision and electrocautery each produced acute allodynia, persistent allodynia was only observed after electrocautery. Under these conditions, we found that the mRNA levels of Small proline rich protein 1A (Sprr1a) and Annexin A10 (Anxa10), which are the key modulators of neuropathic pain, in the spinal cord were more potently and persistently increased by electrocautery than by incision. Furthermore, these genes were overexpressed almost exclusively in chronic postsurgical pain-activated neurons. This event was associated with decreased levels of tri-methylated histone H3 at Lys27 and increased levels of acetylated histone H3 at Lys27 at their promoter regions. On the other hand, persistent allodynia and overexpression of Sprr1a and Anxa10 after electrocautery were dramatically suppressed by systemic administration of GSK-J4, which is a selective H3K27 demethylase inhibitor. These results suggest that the effects of electrocautery contribute to CPSP along with synaptic plasticity and epigenetic modification.

Abstract P363: Annexin-A1 Deficiency Exaggerates Cardiac Remodeling In Angiotensin II-induced Hypertension

Introduction: We have previously demonstrated that the naturally-occurring anti-inflammatory and pro-resolving protein Annexin-A1 (Anx-A1) limits the acute inflammatory response post myocardial infarction, but its impact on chronic inflammation, such as hypertension, has not been explored. This study aims to investigate the role of Anx-A1 in a preclinical model of hypertension, induced by angiotensin-II (Ang-II). Methods: 15-week-old male C57BL/6 or ANXA1 -/- were anesthetized (isoflurane, 2-4% v/v) and implanted with an osmotic minipump randomly assigned to receive Ang-II (0.7mg/kg/day) or vehicle (saline). Radiotelemetry recordings of blood pressure were taken at 10 intermittent timepoints from baseline to the end of the 29-day infusion period. Animals were euthanized with pentobarbitone (100mg/kg; i.p.) at endpoint and organ weights recorded and normalized to bodyweight. Left ventricle (LV) samples were stained with picrosirius red to assess total LV collagen deposition. Results: Ang II-induced mice at the end of the study had elevated mean arterial pressure (MAP), cardiac hypertrophy and fibrosis compared to normotensive mice (Table). Anx-A1 deficient mice given Ang II had an even greater increase in MAP and cardiac remodeling compared to WT. Interestingly, MAP of Anx-A1 deficient mice at baseline is significantly higher compare to C57BL/6 counterparts (Table). Conclusion: This is the first study to demonstrate that deficiency of Anx-A1 exaggerates cardiac remodeling in AngII-induced hypertension, suggesting that endogenous Anx-A1 might play previously unappreciated physiological role in regulating blood pressure. This supports the development of Anx-A1 based pharmacotherapy against hypertension-induced cardiac damage.

(20S) Ginsenoside Rh2 Inhibits STAT3/VEGF Signaling by Targeting Annexin A2

Signal transducers and activators of transcription 3 (STAT3) acts as a transcriptional signal transducer, converting cytokine stimulation into specific gene expression. In tumor cells, aberrant activation of the tyrosine kinase pathway leads to excessive and continuous activation of STAT3, which provides further signals for tumor cell growth and surrounding angiogenesis. In this process, the tumor-associated protein Annexin A2 interacts with STAT3 and promotes Tyr705 phosphorylation and STAT3 transcriptional activation. In this study, we found that (20S) ginsenoside Rh2 (G-Rh2), a natural compound inhibitor of Annexin A2, inhibited STAT3 activity in HepG2 cells. (20S) G-Rh2 interfered with the interaction between Annexin A2 and STAT3, and inhibited Tyr705 phosphorylation and subsequent transcriptional activity. The inhibitory activity of STAT3 leaded to the negative regulation of the four VEGFs, which significantly reduced the enhanced growth and migration ability of HUVECs in co-culture system. In addition, (20S)G-Rh2 failed to inhibit STAT3 activity in cells overexpressing (20S)G-Rh2 binding-deficient Annexin A2-K301A mutant, further proving Annexin A2-mediated inhibition of STAT3 by (20S)G-Rh2. These results indicate that (20S)G-Rh2 is a potent inhibitor of STAT3, predicting the potential activity of (20S)G-Rh2 in targeted therapy applications.

Bufalin induces mitochondrial dysfunction and promotes apoptosis of glioma cells by regulating Annexin A2 and DRP1 protein expression

BackgroundGlioma is a common primary central nervous system tumour, and therapeutic drugs that can effectively improve the survival rate of patients in the clinic are lacking. Bufalin is effective in treating various tumours, but the mechanism by which it promotes the apoptosis of glioma cells is unclear. The aim of this study was to investigate the drug targets of bufalin in glioma cells and to clarify the apoptotic mechanism.MethodsCell viability and proliferation were evaluated by CCK-8 and colony formation assays. Then, the cell cycle and apoptosis, intracellular ion homeostasis, oxidative stress levels and mitochondrial damage were assessed after bufalin treatment. DARTS-PAGE technology was employed and LC–MS/MS was performed to explore the drug targets of bufalin in U251 cells. Molecular docking and western blotting were performed to identify potential targets. siRNA targeting Annexin A2 and the DRP1 protein inhibitor Mdivi-1 were used to confirm the targets of bufalin.ResultsBufalin upregulated the expression of cytochrome C, cleaved caspase 3, p-Chk1 and p-p53 proteins to induce U251 cell apoptosis and cycle arrest in the S phase. Bufalin also induced oxidative stress in U251 cells, destroyed intracellular ion homeostasis, and caused mitochondrial damage. The expression of mitochondrial division-/fusion-related proteins in U251 cells was abnormal, the Annexin A2 and DRP1 proteins were translocated from the cytoplasm to mitochondria, and the MFN2 protein was released from mitochondria into the cytoplasm after bufalin treatment, disrupting the mitochondrial division/fusion balance in U251 cells.ConclusionsOur research indicated that bufalin can cause Annexin A2 and DRP1 oligomerization on the surface of mitochondria and disrupt the mitochondrial division/fusion balance to induce U251 cell apoptosis.Graphic

P–427 Maternal and fetal thrombophilia mutations and the ANXA5 M2 haplotype are more prevalent in first trimester miscarriages of euploid embryos

Abstract Study question Is there a cumulative effect of shared maternal and fetal thrombophilia mutations and the ANXA M2 haplotype in first trimester miscarriages and are there shared genotypes that may predict outcome? Summary answer Coexistence of MTHFR–677CT and PAI–1–4G/5G-mutations in euploid-fetus-mother -pairs may adversely affect early development.Maternal FII–20210GA and PAI–14G mutation were more prevalent in women who miscarried euploid-fetuses. What is known already Spontaneous abortion (SA) is a significant clinical problem with several different etiologies. Certain thrombophilia and folate-related gene mutations have been associated with an increased risk of miscarriage which may be due to impaired coagulation homeostasis. The M2-haplotype of ANXA5-gene promotor encoding the anticoagulation protein Annexin-A5 has also been implicated in early and recurrent SA. However, one important deficiency in the majority of the studies on this subject is a lack of information on the fetal/embryonic contribution to the thrombotic events during placentation and early embryo development. The purpose of this study was to evaluate the prevalence of specific and cumulative thrombophilia mutations in women experiencing first trimester (T1) miscarriages and in their fetal products of conception. Study design, size, duration This is a single Centre retrospective cohort study where we analyzed the frequency and cumulative effect of mutations in 10 thrombophilia/folate related genes and the ANXA5 M2 haplotype in first trimester miscarriages from 229 mother-product of conception (POC) pairs from Jan. 2018-Nov. 2020. Participants/materials, setting, methods All 229 POC DNA samples were confirmed to be fetal and had aneuploidy screening with NGS. We analyzed 10 different mutations in thrombophilia- and folate-related genes (Factor V-Leiden G1691A, Factor V-H1299R, Factor II-G20210A, Factor XIII-V34L, PAI-I–675 4G/5G, FGB–455G/A, MTHFR-C677T and -A1298C, MTR-A2756G, and MTRR- A66G) using single base sequencing methodology and the M2 haplotype of ANXA5 promoter by Sanger sequencing. Maternal–euploid fetus pairs were compared to maternal-aneuploid fetus pairs. SSPS software was used for statistical analyses, and p < 0.05 with CI 95% was considered significant. Main results and the role of chance Aneuploidy was detected in 40.6% (93/229) of POC, and the rest were euploid. Women with euploid fetuses had a significantly higher FII20210GA mutation frequency than those who had aneuploid fetuses (p = 0.02). However, this difference was not detected in the fetuses. Minor allele frequency and genotype frequencies of MTHFR A1298C, MTR A2756G and MTRR A66G mutations as well as for Factor V-Leiden G1691A, Factor V-H1299R and mutations were similar between the groups studied. The frequency of the M2 ANXA5 haplotype was similar between the two groups (3.33% maternal-euploid fetus-pairs vs 3.1% in maternal-aneuploid fetus pairs). PAI-I –675 4G/4G genotype was more frequent in women with euploid fetal losses (25%) vs aneuploid (15%) (p = 0.05), but with no-significant difference in euploid (20%) vs aneuploid fetuses (13%). A cumulative effect of maternal and fetal mutations in MTHFR 677CT and PAI–14G was observed in maternal-euploid fetus pairs (p = 0.0011, OR 3.8 95%CI [1.8–11.3], and p = 0.05, OR 1.2 95%CI [1.12–3.6], respectively). Overall mutation load in tested samples was 4.14 mutations±1.5 [ 1.46±0.8-thrombophilia and 2.45±1.16-folate metabolism] and was similar between both groups of fetuses and their mothers. Limitations, reasons for caution The retrospective nature and cohort design limited the generalizability of our results. The comparison group is maternal-aneuploid fetus- pairs as the reason for the miscarriage is the chromosomal abnormality in the fetus. The ethnic background may be a cofounding factor that needs to be evaluated in a larger study which is ongoing in our center. Wider implications of the findings: Our study found a high mutational load in the 10 folate metabolism and thrombophilia genes we studied in the maternal-fetus pairs. Our findings provide evidence that fetal contribution to placental thrombophilia should be considered in cumulative effect estimations of specific maternal mutations in improvement of management of early miscarriages. Trial registration number Not applicable

Anti-ANX A1 Antibody Therapy in MRL/lpr Murine Model of Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a severe autoimmune disease characterized by dysfunction of immune regulation, overproduction of inflammatory cytokines and attack on normal tissues by self-reactive cells and antibodies. The main role in the pathogenesis plays the autoreactive tandem of B-T cells, responsible for lupus progression and acceleration. Both activated B and T cells express a phospholipid binding protein Annexin A1 and abnormal levels of the protein were found in murine and human autoimmune syndromes, potentiating its role as a therapeutic target. Here, using anti-annexin A1 antibody we explore its property to modulate the autoimmune response in MRL/lpr mouse model of lupus. Anti-ANX A1 antibody was tested in vitro using spleen cells from MRL/lpr mice to determine the effect on lymphocyte activation, plasma cells differentiation, apoptosis and proliferation by flow cytometry and ELISpot assays. Subsequently, several groups of young (disease-free) and old (sick) MRL/lpr mice were treated with the antibody to determine the levels of panel auto-antibodies and cytokines, T cell arrest and migration. Treatment of splenocytes with anti-ANX A1 antibody inhibited T-cell activation and proliferation, suppressed anti-dsDNA antibody-producing plasma cells and affected B cell apoptosis. Administration of the antibody to MRL/lpr mice resulted to decreased autoantibody levels to various lupus antigens, suppressed T cell migration from lymph nodes and increased the levels of IL4 mRNA compared to the control group. Anti-ANX A1 antibody therapy suppresses B and T cell over-activation and down- modulates disease activity.

A Novel Multisystem Proteinopathy Caused by a Missense ANXA11 Variant.

Protein misfolding plays a central role not only in amyotrophic lateral sclerosis (ALS), but also in other conditions, such as frontotemporal dementia (FTD), inclusion body myopathy (hIBM) or Paget's disease of bone. The concept of multisystem proteinopathies (MSP) was created to account for those rare families that segregate at least 2 out of these 4 conditions in the same pedigree. The calcium-dependent phospholipid-binding protein annexin A11 was recently associated to ALS in European pedigrees. Herein, we describe in detail 3 Brazilian families presenting hIBM (isolated or in combination with ALS/FTD) caused by the novel p.D40Y change in the gene encoding annexin A11 (ANXA11). We collected clinical, genetic, pathological and skeletal muscle imaging from 11 affected subjects. Neuroimaging was also obtained from 8 patients and 8 matched controls. Clinico-radiological phenotype of this novel hIBM reveals a slowly progressive predominant limb-girdle syndrome, but with frequent axial (ptosis/dropped head) and distal (medial gastrocnemius) involvement as well. Muscle pathology identified numerous rimmed vacuoles with positive annexin A11, TDP-43 and p62 inclusions, but no inflammation. Central nervous system was also involved: two patients had FTD, but diffusion tensor imaging uncovered multiple areas of cerebral white matter damage in the whole group (including the corticospinal tracts and frontal subcortical regions). These findings expand the phenotypic spectrum related to ANXA11. This gene should be considered the cause of a novel multisystem proteinopathy (MSP type 6), rather than just ALS. ANN NEUROL 2021;90:239-252.

AnnexinA5 Might Suppress the Phenotype of Human Gastric Cancer Cells via ERK Pathway.

Gastric cancer is one of the most fatal diseases around the world. However, the mechanism of the development of gastric cancer is still not clarified. In addition, the anticancer drugs have cytotoxicity with different degrees. AnnexinA5, a member of the annexin family, has a great binding ability with the membrane phospholipid in a calcium dependent manner and is involved in the development of various cancers. This study aims to explore the influence of annexinA5 on human gastric cancer cells and whether it has the potential to be an auxiliary treatment to gastric cancer. In this study, the role of annexinA5 was detected from both the endogenous and the exogenous aspects on the gastric cancer cell lines MGC-803 and MKN-45. The cells were divided into a knockdown group in which RNA interference technique was used to suppress annexinA5 expression and a protein-supplementing group in which annexinA5 protein was added in the culture supernatant. After the suppression ratio of RNA interference was determined and the IC50 of annexinA5 protein was decided respectively, the cells’ proliferation was detected by MTT assay, colony formation assay, and the expression of PCNA. FCM assay and PI staining methods were applied to test cell apoptosis and necrosis. To investigate whether ANXA5 influence cell metastasis, wound healing assay and transwell assay were employed. To further detect the mechanism of annexinA5 action, the signal pathway was examined with Western Blot method. When ANXA5 gene was knocked down, cell proliferation and metastasis were promoted, while cell apoptosis was suppressed. On the other hand, after the annexinA5 protein was applied to the gastric cancer cells, cell proliferation and metastasis were inhibited, while cell apoptosis and necrosis were promoted. AnnexinA5 played its role via ERK signal pathway. ANXA5 acted as tumor suppressor gene in the gastric cancer by suppressing ERK signal pathway and has the potentiality to be an auxiliary anticancer agent.

Protein Signatures of Parathyroid Carcinomas Using Proteomic Analyses

Parathyroid carcinomas are rare endocrine tumors derived from the parathyroid glands with poor prognosis. Moreover, parathyroid carcinomas are resistant to radiation or drug therapy, and surgical resection is the only treatment option. Understanding the molecular pathogenesis of parathyroid carcinomas may pave the way for early diagnostic biomarkers and therapeutic targets. Therefore, we aimed to elucidate the protein signatures for parathyroid carcinomas through quantitative proteomic analyses. We performed liquid chromatography with tandem mass spectrometry (LC-MS/MS) technique with formalin-fixed paraffin-embedded (FFPE) samples and reached a quantitative depth of more than 5,000 proteins per sample. For the analyses, 23 parathyroid carcinoma and 15 adenoma samples were collected from five tertiary hospitals in Korea. Patients’ mean age was 52 years, and 24 (63%) were female. Patients with parathyroid carcinoma had higher parathyroid hormone (PTH) and serum calcium level than adenomas (PTH, 1077.6 ± 760.7, 181.8 ± 139.8 pg/mL; calcium 13.0 ± 2.8, 11.3 ± 1.0 mg/dL, respectively). From the proteomic expression profiling, there were 137 differentially expressed proteins with the cutoff of both p <0.05 and fold change > 1.5. Using the Ingenuity Pathway Analysis (IPA), top enriched canonical pathways in parathyroid carcinomas included glycoprotein-6 signaling related to the coagulation pathway, acute phase response, mTOR, and clathrin-/caveolar-mediated endocytosis signaling. In transcription factor analysis, TGFβ and TP53 were activated in carcinoma, and these factors were up-regulators of CD44 antigen and Annexin A2 (ANXA2) proteins. In network analysis, α-1-acid glycoprotein 1 (ORM1), laminin subunit β-2 (LAMB2), and Serpin family (SERPIN) proteins were derived as essential proteins and correlated to the AKT complex. Also, with the support vector machine (SVM)-based classification method, we derived a set of proteins that can discriminate carcinomas from adenomas, which consists of Carbonic anhydrase 4 (CA4), α/β hydrolase domain-containing protein 14B (ABHD14B), CD44, LAMB2, phosphatidylinositol transfer protein β isoform (PITPNB), and ORM1, with the lowest error rate of 11.1%. In conclusion, from the proteomics analyses of parathyroid neoplasms, newly recognized pathways - signaling related to coagulation, acute phase response, and endocytosis - were enriched in parathyroid carcinoma in addition to the known mTOR signaling pathway. The proteins such as α-1-acid glycoprotein and laminin subunit β-2 from SVM classification and network analyses could be the distinctive signature of carcinoma and may provide insights into the therapeutic target.

Human colorectal cancer-associated carbohydrate antigen on annexin A2 protein.

Cancer-associated antigens are not only a good marker for monitoring cancer progression but are also useful for molecular target therapy. In this study, we aimed to generate a monoclonal antibody that preferentially reacts with colorectal cancer cells relative to noncancerous gland cells. We prepared antigens composed of HT-29 colorectal cancer cell lysates that were adsorbed by antibodies to sodium butyrate-induced enterocytically differentiated HT-29 cells. Subsequently, we generated a monoclonal antibody, designated 12G5A, which reacted with HT-29 colon cancer cells, but not with sodium butyrate-induced differentiated HT-29 cells. Immunohistochemical staining revealed 12G5A immunoreactivity in all 73 colon cancer tissue specimens examined at various degrees, but little or no immunoreactivity in noncancerous gland cells. Notably, high 12G5A immunoreactivity, which was determined as more than 50% of colon cancer cells intensively stained with 12G5A antibody, exhibited significantly higher association with a poor overall survival rate of patients with colorectal cancer (P = 0.0196) and unfavorable progression-free survival rate of patients with colorectal cancer (P = 0.0418). Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, si-RNA silencing analysis, enzymatic deglycosylation, and tunicamycin treatment revealed that 12G5A recognized the glycosylated epitope on annexin A2 protein. Our findings indicate that 12G5A identified a cancer-associated glycosylation epitope on annexin A2, whose expression was related to unfavorable colorectal cancer behavior. KEY MESSAGE: • 12G5A monoclonal antibody recognized a colorectal cancer-associated epitope. • 12G5A antibody recognized the N-linked glycosylation epitope on annexin A2. • 12G5A immunoreactivity was related to unfavorable colorectal cancer behavior.

Pro-Resolving Mediator Annexin A1 Regulates Intracellular Ca2+ and Mucin Secretion in Cultured Goblet Cells Suggesting a New Use in Inflammatory Conjunctival Diseases.

The amount of mucin secreted by conjunctival goblet cells is regulated to ensure the optimal level for protection of the ocular surface. Under physiological conditions lipid specialized pro-resolving mediators (SPM) are essential for maintaining tissue homeostasis including the conjunctiva. The protein Annexin A1 (AnxA1) can act as an SPM. We used cultured rat conjunctival goblet cells to determine if AnxA1 stimulates an increase in intracellular [Ca2+] ([Ca2+]i) and mucin secretion and to identify the signaling pathways. The increase in [Ca2+]i was determined using fura2/AM and mucin secretion was measured using an enzyme-linked lectin assay. AnxA1 stimulated an increase in [Ca2+]i and mucin secretion that was blocked by the cell-permeant Ca2+ chelator BAPTA/AM and the ALX/FPR2 receptor inhibitor BOC2. AnxA1 increased [Ca2+]i to a similar extent as the SPMs lipoxin A4 and Resolvin (Rv) D1 and histamine. The AnxA1 increase in [Ca2+]i and mucin secretion were inhibited by blocking the phospholipase C (PLC) pathway including PLC, the IP3 receptor, the Ca2+/ATPase that causes the intracellular Ca2+ stores to empty, and blockade of Ca2+ influx. Inhibition of protein kinase C (PKC) and Ca2+/calmodulin-dependent protein kinase also decreased the AnxA1-stimulated increase in [Ca2+]i and mucin secretion. In contrast inhibitors of ERK 1/2, phospholipase A2 (PLA2), and phospholipase D (PLD) did not alter AnxA1-stimulated increase in [Ca2+]i, but did inhibit mucin secretion. Activation of protein kinase A did not decrease either the AnxA1-stimulated rise in [Ca2+]i or secretion. We conclude that in health, AnxA1 contributes to the mucin layer of the tear film and ocular surface homeostasis by activating the PLC signaling pathway to increase [Ca2+]i and stimulate mucin secretion and ERK1/2, PLA2, and PLD to stimulate mucin secretion from conjunctival goblet cells.

A tumorigenic cell line derived from a hamster cholangiocarcinoma associated with Opisthorchis felineus liver fluke infection

AimsThe food-born trematode Opisthorchis felineus colonizes bile ducts of the liver of fish-eating mammals including humans. There is growing evidence that this liver fluke is a risk factor for cholangiocarcinoma (CCA). Cancer cell lines are necessary for drug screening and for identifying protein markers of CCA. The aim was to establish a cell line derived from cholangiocarcinoma associated with opisthorchiasis felinea. Main methodsAllotransplantation, immunohistochemistry, karyotype analysis, cell culture techniques, immunocytochemistry and real-time PCR. Key findingsHere we repot the establishment of first CCA cell line, CCA-OF, from a primary tumor of an experimental CCA in Syrian hamsters treated with low doses of dimethyl nitrosamine and associated with O. felineus infection. The cell line was found to be allotransplantable. Expression of epithelial and mesenchymal markers (cytokeratin 7, glycosyltransferase exostosin 1, Ca2+-dependent phospholipid-binding protein annexin A1 and vimentin) was demonstrated by immunostaining of the primary tumors, CCA-OF cells, and allotransplants. CCA-OF cells were found to express presumed CCA biomarkers previously detected in both human and experimental tumors associated with the liver fluke infection. The cells were diploid-like (2n = 42–46) with complex chromosomal rearrangements and have morphological features of epithelial-like cells. The usefulness of the CCA-OF cell model for antitumor activity testing was demonstrated by an analysis of effects of resveratrol treatment. It was shown that resveratrol treatment inhibited the proliferation and the migration ability of CCA-OF cells. SignificanceThus, the allotransplantable CCA-OF cell line can be used in studies on helminth-associated cholangiocarcinogenesis and for the testing of antitumor drugs.

HDX-MS for Epitope Characterization of a Therapeutic ANTIBODY Candidate on the Calcium-Binding Protein Annexin-A1.

Annexin-A1 (ANXA1) belongs to a class of highly homologous Ca2+-dependent phospholipid-binding proteins. Its structure consists of a core region composed of four homologous repeats arranged in a compact, hydrolysis-resistant structure and an N-terminal region with a Ca2+-dependent conformation. ANXA1 is involved in several processes, including cell proliferation, apoptosis, metastasis, and the inflammatory response. Therefore, the development of antibodies blocking selected regions on ANXA1 holds great potential for the development of novel therapeutics treating inflammatory and cancer diseases. Here, we report the interaction site between an ANXA1-specific antibody known to inhibit T cell activation without adverse cytotoxic effects and ANXA1 using amide hydrogen–deuterium exchange mass spectrometry (HDX-MS). For the epitope determination, we applied two bottom-up HDX-MS approaches with pepsin digestion in solution and immobilized on beads. Both strategies revealed the interaction region within domain III of ANXA1 in Ca2+-bound conformation. The antibody-binding region correlates with the hydrophobic binding pocket of the N-terminal domain formed in the absence of calcium. This study demonstrates that even cryptic and flexible binding regions can be studied by HDX-MS, allowing a fast and efficient determination of the binding sites of antibodies which will help to define a mode of action profile for their use in therapy.

Quantitative Proteomics Analysis for the Identification of Differential Protein Expression in Calf Muscles between Young and Old SD Rats Using Mass Spectrometry.

Aging is associated with loss of muscle mass and strength that leads to a condition termed sarcopenia. Impaired conditions, morbidity, and malnutrition are the factors of devaluation of muscle fibers in aged animals. Satellite cells play an important role in maintaining muscle homeostasis during tissue regeneration and repair. Proteomic profiling on the skeletal muscle tissues of different age group rats helps to determine the differentially expressed (DE) proteins, which may eventually lead to the development of biomarkers in treating the conditions of sarcopenia. In this study, nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS) analysis was implemented in the calf tissues of young and old groups of rats. The mass spectrometry (MS) analysis revealed the presence of 335 differentially expressed proteins between the two different age conditions, among which those based on log-fold change 25 proteins were upregulated and 77 were downregulated. The protein–protein interaction network analysis revealed 18 upregulated proteins with three distinct interconnected networks and 57 downregulated proteins with two networks. Further, gene ontology (GO) enrichment analysis showed the biological process, cellular component, and molecular function of the differential proteins. Pathway enrichment analysis of the DE proteins identified nine significantly enriched pathways with a list of eight significant genes (Cryab, Hspb2, Acat1, Ak1, Adssl1, Anxa5, Gys1, Ogdh, Gc, and Adssl1). Quantification of significant genes by quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the downregulation at the mRNA level. Western blot analysis of their protein expression showed concordant results on two candidate proteins (Ogdh and annexin 5) confirming their differential regulation between the two age groups of rats. Thus, these proteomic approaches on young and aged rats provide insights into the development of protein targets in the treatment of sarcopenia (muscle loss).