Formylpeptide receptors in GtoPdb v.2021.2

The formylpeptide receptors (nomenclature agreed by the NC-IUPHAR Subcommittee on the formylpeptide receptor family [196]) respond to exogenous ligands such as the bacterial product fMet-Leu-Phe (fMLP) and endogenous ligands such as lipoxin A4 (LXA4), 15-epi-lipoxin A4, annexin I , cathepsin G, amyloid β42, serum amyloid A and spinorphin, derived from β-haemoglobin. FPR1 also serves as a plague receptor for selective destruction of human immune cells by Y. pestis [135]. The FPR1/2 agonists 'compound 17b' and 'compound 43' have shown cardiac protective functions [149, 64].

Affinity interactions of Actin with HS1 and Annexin I, II, IV, V and VI in RAW264.7 macrophages in vitro and in the Fc-receptor complex

Actin binding proteins HS1 and Annexin I, II, III, IV, V and VI proteins were examined in RAW macrophages in vitro using affinity chromatography, LC-MS/MS, western blot, cell staining and expression of fluorescent proteins. Actins and many related actin isoforms were observed in the phagosome. However, actins were also observed on polystyrene beads incubated with crude extracts readily bound to NHS affinity chromatography beads in the absence of anti-actin antibodies. Annexin II and V and the haematopoietic specific proteins HS1 (also called Lyn substrate) were prominently observed in isolated phagosomes and not observed in control beads incubated with crude extracts of RAW macrophages. Annexin II and V as well as G3BP, FLT1, FARP2, STARD13, RAB25, and FRAP1 were observed to bind actin in affinity chromatography experiments using LC-MS/MS and western blot. Cell staining and/or expression of GFP constructs showed that actin, HS1 and Annexin V prominently accumulate at the nascent Fc-receptor complex of RAW macrophages.

Affinity interactions of Actin with HS1 and Annexin I, II, IV, V and VI in RAW264.7 macrophages in vitro and in the Fc-receptor complex

Actin binding proteins HS1 and Annexin I, II, III, IV, V and VI proteins were examined in RAW macrophages in vitro using affinity chromatography, LC-MS/MS, western blot, cell staining and expression of fluorescent proteins. Actins and many related actin isoforms were observed in the phagosome. However, actins were also observed on polystyrene beads incubated with crude extracts readily bound to NHS affinity chromatography beads in the absence of anti-actin antibodies. Annexin II and V and the haematopoietic specific proteins HS1 (also called Lyn substrate) were prominently observed in isolated phagosomes and not observed in control beads incubated with crude extracts of RAW macrophages. Annexin II and V as well as G3BP, FLT1, FARP2, STARD13, RAB25, and FRAP1 were observed to bind actin in affinity chromatography experiments using LC-MS/MS and western blot. Cell staining and/or expression of GFP constructs showed that actin, HS1 and Annexin V prominently accumulate at the nascent Fc-receptor complex of RAW macrophages.

Proteomic Analysis of Differentially Expressed Proteins in Endothelial Cells Under Low Shear Stress

Background: Shear stress (SS) affects the morphology, proliferation, differentiation and migration of endothelial cells, and regulates protein expression.Methods: This study aims to conduct a proteomic analysis of human umbilical vein endothelial cells (HUVECs) under low SS. HUVECs were cultured on glass slides (test group n=30; control group n=30) and transferred to parallel-plate flow chamber. The test group was subjected to low SS at 4.58 dyne/cm2 for 2 hours, and the control group was maintained under static conditions. Approximately 2×107 cells were collected from each group and analyzed by two-dimensional electrophoresis. The protein from each spot were identified by mass spectrometry. The biological functions and processes associated with the identified proteins were evaluated using the NCBInr, SwissProt, and DAVID databases. The protein expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, filamin B, Ras-related nuclear protein (Ran), and talin were assessed by Western blotting.Results: A total of 234 proteins were analyzed; 14 proteins were upregulated and 78 were downregulated in the test group compared with the control group. The expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, and filamin B was significantly decreased, whereas the expression of Ran and talin was significantly increased in the test group.Conclusion: The current results provide evidence that SS changes the protein profile of HUVECs.

A knock-down cell-based study for the functional analysis of chloride intracellular channel 1 (CLIC1): Integrated proteomics and microarray study

Background: Previously, we detected that chloride intracellular channel 1 (CLIC1) was involved in the pathogenesis of atopic dermatitis (AD). Objective: In this study, we aimed to use high-throughput screening (HTS) approaches to identify critical factors associated with the function of CLIC1 in knock-down cells. Methods: We down-regulated CLIC1 in human A549 cells via siRNA and then conducted serial HTS studies, including proteomics integrated with a microarray and the implementation of bioinformatics algorithms. Results: Together, these approaches identified several important proteins and genes associated with the function of CLIC1. These proteins and genes included tumor rejection antigen (gp96) 1, nucleophosmin, annexin I, keratin 1 and 10, FLNA protein, enolase 1, and metalloprotease 1, which were found using two-dimensional electrophoresis (2-DE) proteomics. Separately, NTNG1, SEMA5A, CLEC3A, GRPR, GNGT2, GRM5, GRM7, DNMT3B, CXCR5, CCL11, CD86, IL2, MNDA, TLR5, IL23R, DPP6, DLGAP1, CAT, GSTA1, GSTA2, GSTA5, CYP2E1, ADH1A, ESR1, ARRDC3, A1F1, CCL5, CASP8, DNTT, SQSTM1, PCYT1A, and SLCO4C1 were found using a DNA microarray integrated with PPI mapping. Conclusion: CCL11 is thought to be a particularly critical gene among the candidate genes detected in this study. By integrating the datasets and utilizing the strengths of HTS, we obtained new insights into the functional role of CLIC1, including the use of CLIC1-associated applications in the treatment of human diseases such as AD.

Proteomic analysis of differentially expressed proteins in endothelial cells under low shear stress

Background: Shear stress (SS) affects the morphology, proliferation, differentiation and migration of endothelial cells, and regulates protein expression.Methods: This study aims to conduct a proteomic analysis of human umbilical vein endothelial cells (HUVECs) under low SS. HUVECs were cultured on glass slides (test group n=30; control group n=30) and transferred to parallel-plate flow chamber. The test group was subjected to low SS at 4.58 dyne/cm2 for 2 hours, and the control group was maintained under static conditions. Approximately 2´107 cells were collected from each group and analyzed by two-dimensional electrophoresis. The protein from each spot were identified by mass spectrometry. The biological functions and processes associated with the identified proteins were evaluated using the NCBInr, SwissProt, and DAVID databases. The protein expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, filamin B, Ras-related nuclear protein (Ran), and talin were assessed by Western blotting.Results: A total of 234 proteins were analyzed; 14 proteins were upregulated and 78 were downregulated in the test group compared with the control group. The expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, and filamin B was significantly decreased, whereas the expression of Ran and talin was significantly increased in the test group.Conclusion: The current results provide evidence that SS changes the protein profile of HUVECs.

Formylpeptide receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The formylpeptide receptors (nomenclature agreed by the NC-IUPHAR Subcommittee on the formylpeptide receptor family [185]) respond to exogenous ligands such as the bacterial product fMet-Leu-Phe (fMLP) and endogenous ligands such as annexin I , cathepsin G, amyloid β42, serum amyloid A and spinorphin, derived from β-haemoglobin.

Proteomic analysis of differentially expressed proteins in endothelial cells under low shear stress

Shear stress (SS) affects the morphology, migration, differentiation, and proliferation of endothelial cells, and regulates protein expression. The objective of this study was to perform a proteomic analysis of human umbilical vein endothelial cells (HUVECs) under low SS. HUVEC lines were cultured on glass slides (test group n=30; control group n=30) and transferred to a parallel-plate flow chamber. The test group was subjected to low SS at 4.58 dyne/cm2 for 2 hours, and the control group was maintained under static conditions. Approximately 2×107 cells were collected from each group and analyzed by two-dimensional electrophoresis. The proteins from each spot were identified by mass spectrometry. The biological functions and processes associated with the identified proteins were evaluated using the NCBInr, SwissProt, and DAVID databases. The protein expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, filamin B, Ras-related nuclear protein (Ran), and talin were assessed by Western blotting. A total of 234 proteins were analyzed; 14 proteins were upregulated and 78 were downregulated in the test group compared with the control group. The expression of 14-3-3 proteins, annexin I, annexin II, zyxin, filamin A, and filamin B was significantly decreased, whereas the expression of Ran and talin was significantly increased in the test group. It is known that SS induces the remodeling of endothelial cells by modulating protein expression. However, the mechanisms underlying this modulation are unknown. The present results provide evidence that SS changes the protein profile of HUVECs.

High Risk Acute Lymphoblastic Leukemia with Rapid NOD/SCID Engraftment Is Characterized by High Protein Expression of CYCLIN B, Beta-CATENIN, ANNEXIN I and Decreased PKC Alpha Activation

Abstract 1457 Acute lymphoblastic leukemia (ALL) is the most frequent form of childhood cancer. Although therapy efforts have achieved cure rates of above 80%, about every fifth patient encounters relapse, which is associated with poor outcome. Risk stratification based on prognostic factors is critical for selecting appropriate treatment intensity to improve therapy efficacy and reduce toxicity. However, in spite of new strategies for risk stratification, the majority of relapsed patients are initially stratified into non-high risk groups pointing out the need to identify new prognostic markers that might refer to novel therapeutic targets. We have recently shown in a NOD/SCID/huALL mouse xenotranplant model that early relapse of pediatric B-cell precursor (BCP-) ALL patients is reflected by rapid engraftment (time to leukemia/ TTLshort) of primary ALL cells. In this study we aimed to identify differently expressed and activated proteins in xenograft ALL of distinct engraftment subgroups employing a Reverse Phase Protein Array (RPPA) strategy. Levels of total or activated proteins were analyzed in patient derived xenograft BCP-ALL samples characterized with respect to their NOD/SCID engraftment phenotype (TTLshort n= 7, TTLlong n= 9). The proteome of each sample was immobilized on nitrocellulose coated glass slides and overall protein expression or phosphorylation of 51 key signaling molecules was detected by incubation with the corresponding specific antibodies. Protein expression and/ or activation was quantified and compared between the different engraftment subgroups (TTLshort/ early relapse versus TTLlong/ good prognosis). RPPA results were validated by Western Blot analyses. The association of NOD/SCID/huALL engraftment and clinical patient outcome was analyzed and revealed a significantly inferior survival of patients with the TTLshort in contrast to the TTLlong phenotype also in this subgroup of patients (Kaplan Meier, log rank, P=.003). Upon comparison of the protein expression data (Shrinkage t-test, P <.05, fold change ≥ 1.5) in the TTL subgroups the three proteins CYCLIN B, beta-CATENIN and ANNEXIN I were identified to be overexpressed in TTLshort. In addition, an increased activation of protein-kinase C alpha (phosporylated serine 657, p-PKC alpha S657) was detected in TTLlong leukemia samples. CYCLIN B is a positive regulator of the cell cycle and highly expressed in the G2/M phase. Consistent with its pro-proliferative function, CYCLIN B was identified to be up-regulated in leukemia samples with rapid NOD/SCID/huALL growth (TTLshort/ early relapse phenotype) (P=.013). Interestingly, the level of CYCLIN B protein expression correlates negatively to TTL (Spearman, rs=-.516, P=.041), suggesting a direct association of leukemia cell cycle progression and leukemia engraftment in the NOD/SCID/huALL xenograft model. Beta-CATENIN, a molecule involved in WNT-signaling and cell adhesion but also associated with apoptosis inhibition and cell growth in other hematological malignancies, was found to be over-expressed in TTLshort (P=.006). Furthermore, beta-CATENIN protein levels were reversely correlated to TTL (Spearman, rs=-.557, P=.025). Additionally, ANNEXIN I, involved in the regulation of inflammation and apoptosis and reported to be up-regulated in hairy cell leukemia, showed high protein levels in TTLshort (P=.033). Protein-kinase C alpha was previously reported to induce apoptosis in an ALL- cell line and showed increased activation in non-relapsing pediatric T-ALL patient samples. In line with this, we found higher levels of PKC alpha activation in the TTLlong -group (P= 8.5e–9), indicating an association with good prognosis. Taken together, this study identified differentially expressed proteins in prognostic subgroups of BCP-ALL patients with distinct clinical outcomes, which can be further evaluated as new prognostic markers and therapeutic targets. Disclosures: Debatin: Apogenix GmbH: Patents & Royalties, Research Funding.