FurinDB (freely available online at http://www.nuolan.net/substrates.html) is a database of furin substrates. This database includes experimentally verified furin cleavage sites, substrates, species, experimental methods, original publications of experiments and associated drugs targeting furin substrates. The current database release contains 126 furin cleavage sites from three species: mammals, bacteria and viruses. A main feature of this database is that all furin cleavage sites are recorded as a 20-residue motif, including one core region (eight amino acids, P6–P2’) and two flanking solvent accessible regions (eight amino acids, P7–P14, and four amino acids, P3’–P6’), that represent our current understanding of the molecular biology of furin cleavage. This database is important for understanding the molecular evolution and relationships between sequence motifs, 3D structures, cellular functions and physical properties required by furin for cleavage, and for elucidating the molecular mechanisms and the progression of furin cleavage associated human diseases, including pathogenic infections, neurological disorders, tumorigenesis, tumor invasion, angiogenesis, and metastasis. FurinDB database will be a solid addition to the publicly available infrastructure for scientists in the field of molecular biology.

Furin cleaves diverse types of protein precursors in the secretory pathway. The substrates for furin cleavage possess a specific 20-residue recognition sequence motif. In this report, based on the functional characterisation of the 20-residue sequence motif, we developed a furin cleavage site prediction tool, PiTou, using a hybrid method composed of a hidden Markov model and biological knowledge-based cumulative probability score functions. PiTou can accurately predict the presence and location of furin cleavage sites in protein sequences with high sensitivity (96.9%) and high specificity (97.3%). PiTou's prediction scores are biological meaningful and reflect binding strength and solvent accessibility of furin substrates. A prediction result is interpreted within cellular contexts: subcellular localisation, cellular function and interference by other dynamic protein modifications. Combining next-generation sequencing, PiTou can help with elucidating the molecular mechanism of furin cleavage-associated human diseases. PiTou has been made freely available at the associated website.

Integrin α_vβ₃ interacting with the short Arg-Gly-Asp (RGD) motif plays a critical role in the progression of several types of tumors. However, the effects of the RGD structure (cyclic or linear) with integrin α_vβ₃ at the atomic level remain poorly understood. Here, we performed association and dissociation dynamic simulations for integrin α_vβ₃ in complex with a linear or cyclic pentapeptide by steered molecular dynamics simulations. Compared with cyclic RGD, the linear RGD peptide triggers instability of the configurational changes, mainly resting with the RGD domain due to its flexibility. The main interaction energy between Mg²⁺ and cyclic RGD is much stronger than that of the linear RGD system by the well shield to lessen attacks by free water molecules. The force-dependent dissociation results show that it is easier for linear RGD peptides to leave the active site and much quicker than the cyclic RGD ligand, whereas it is harder to enter the appropriate active binding site in linear RGD. The Ser¹²³-Asp^RGD bond may play a critical role in the allosteric pathway. Our findings provide insights into the dynamics of α_vβ₃ interactions with linear and cyclic RGD ligands and contribute to the application of RGD-based strategies in preclinical therapy.

Activation of integrins is crucial for recruitment of flowing leukocytes to inflammatory or injured vascular sites, but their spatiotemporal characteristics are incompletely understood. We discovered that β₂-integrin activation over the entire surface of neutrophils on immobilized P-selectin occurred via mitogen-activated protein kinase (MAPK) or non-MAPK signaling with a minute-level timescale in a force-dependent manner. In flow, MAPK signaling required intracellular Ca²⁺ release to activate integrin within 2 min. Integrin activation via non-MAPK signaling occurred first locally in the vicinity of ligated P-selectin glycoprotein ligand-1 (PSGL-1) within sub-seconds, and then over the entire cell surface within 1 min in an extracellular Ca²⁺ influx-dependent manner. The transition from a local (but rapid) to global (but slow) activation mode was triggered by ligating the freshly activated integrin. Lipid rafts, moesin, actin, and talin were involved in non-MAPK signaling. Fluid loads had a slight effect on local integrin activation with a second-level timescale, but served as enhancers of global integrin activation.

P-selectin engagement of P-selectin glycoprotein ligand-1 (PSGL-1) causes circulating leukocytes to roll on and adhere to the vascular surface, and mediates intracellular calcium flux, a key but unclear event for subsequent arresting firmly at and migrating into the infection or injured tissue. Using a parallel plate flow chamber technique and intracellular calcium ion detector (Fluo-4 AM), the intracellular calcium flux of firmly adhered neutrophils on immobilized P-selectin in the absence of chemokines at various wall shear stresses was investigated here in real time by fluorescence microscopy. The results demonstrated that P-selectin engagement of PSGL-1 induced the intracellular calcium flux of firmly adhered neutrophils in flow, increasing P-selectin concentration enhanced cellular calcium signaling, and, force triggered, enhanced and quickened the cytoplasmic calcium bursting of neutrophils on immobilized P-selectin. This P-selectin-induced calcium signaling should come from intracellular calcium release rather than extracellular calcium influx, and be along the mechano-chemical signal pathway involving the cytoskeleton, moesin and Spleen tyrosine kinase (Syk). These results provide a novel insight into the mechano-chemical regulation mechanism for P-selectin-induced calcium signaling of neutrophils in flow.