Data on the inhibition of cell proliferation and invasion by the D2A-Ala peptide derived from the urokinase receptor.

The data presented in this article are connected to our research article entitled "D2A-Ala peptide derived from the urokinase receptor exerts anti-tumoural effects in vitro and in vivo" (Furlan et al., 2018). These data further extend our understanding of the inhibitory effects of D2A-Ala peptide. Dose-response curve using a wide range of concentrations of D2A-Ala shows that this peptide has no effects per se on proliferation of rat smooth muscle cells (RSMC). However, D2A-Ala dose-dependently inhibits epidermal growth factor (EGF)-induced RSMC proliferation. Kinetics lasting up to seven days revealed that D2A-Ala peptide completely blocked EGF-promoted RSMC proliferation. Moreover, D2A-Ala peptide inhibited invasion of HT 1080 cells towards RSMC.

D2A-Ala peptide derived from the urokinase receptor exerts anti-tumoural effects in vitro and in vivo.

D2A-Ala is a synthetic peptide that has been created by introducing mutations in the original D2A sequence, 130IQEGEEGRPKDDR142 of human urokinase receptor (uPAR). In vitro, D2A-Ala peptide displays strong anti-tumoural properties inhibiting EGF-induced chemotaxis, invasion and proliferation of a human fibrosarcoma cell line, HT 1080, and a human colorectal adenocarcinoma cell line, HT 29. D2A-Ala exerts its effects by preventing EGF receptor (EGFR) phosphorylation. To test D2A-Ala in vivo, this peptide was PEGylated generating polyethyleneglycol (PEG)-D2A-Ala peptide. PEGylation did not alter the inhibitory properties of D2A-Ala. Human tumour xenografts in the immunodeficient nude mice using HT 1080 and HT 29 cell lines showed that PEG-D2A-Ala significantly prevents tumour growth decreasing size, weight and density of tumours. The most efficient doses of the peptide were 5 and 10 mg/kg, thereby relevant for possible development of the peptide into a drug against cancer in particular tumours expressing EGFR.

D2A sequence of the urokinase receptor induces cell growth through αvß3 integrin and EGFR.

The urokinase receptor (uPAR) stimulates cell proliferation by forming a macromolecular complex with αvß3 integrin and the epidermal growth factor receptor (EGFR, ErbB1 or HER1) that we name the uPAR proliferasome. uPAR transactivates EGFR, which in turn mediates uPAR-initiated mitogenic signal to the cell. EGFR activation and EGFR-dependent cell growth are blocked in the absence of uPAR expression or when uPAR activity is inhibited by antibodies against either uPAR or EGFR. The mitogenic sequence of uPAR corresponds to the D2A motif present in domain 2. NMR analysis revealed that D2A synthetic peptide has a particular three-dimensional structure, which is atypical for short peptides. D2A peptide is as effective as EGF in promoting EGFR phosphorylation and cell proliferation that were inhibited by AG1478, a specific inhibitor of the tyrosine kinase activity of EGFR. Both D2A and EGF failed to induce proliferation of NR6-EGFR-K721A cells expressing a kinase-defective mutant of EGFR. Moreover, D2A peptide and EGF phosphorylate ERK demonstrating the involvement of the MAP kinase signalling pathway. Altogether, this study reveals the importance of sequence D2A of uPAR, and the interdependence of uPAR and EGFR.

CEP120 and SPICE1 cooperate with CPAP in centriole elongation.

Centrosomes organize microtubule (MT) arrays and are comprised of centrioles surrounded by ordered pericentriolar proteins. Centrioles are barrel-shaped structures composed of MTs, and as basal bodies they template the formation of cilia/flagella. Defects in centriole assembly can lead to ciliopathies and genome instability. The assembly of procentrioles requires a set of conserved proteins. It is initiated at the G1-to-S transition by PLK4 and CEP152, which help recruit SASS6 and STIL to the vicinity of the mother centriole to organize the cartwheel. Subsequently, CPAP promotes centriolar MT assembly and elongation in G2. While centriole integrity is maintained by CEP135 and POC1 through MT stabilization, centriole elongation requires POC5 and is restricted by CP110 and CEP97. How strict control of centriole length is achieved remains unclear. Here, we show that CEP120 and SPICE1 are required to localize CEP135 (but not SASS6, STIL, or CPAP) to procentrioles. CEP120 associates with SPICE1 and CPAP, and depletion of any of these proteins results in short procentrioles. Furthermore, CEP120 or CPAP overexpression results in excessive centriole elongation, a process dependent on CEP120, SPICE1, and CPAP. Our findings identify a shared function for these proteins in centriole length control.

The urokinase receptor interactome.

The urokinase receptor (uPAR) was originally identified as the membrane receptor of the serine protease urokinase (uPA), thereby implicated in the plasminogen activation cascade and regulation of pericellular proteolysis. Later on, vitronectin was showed to be another major ligand providing uPAR with a role in cell adhesion. Other unrelated ligands have been subsequently reported including for example factor XII and SRPX2 expanding the functions of uPAR to unexpected biological areas such as the initiation of the coagulation cascade or the regulation of language development. Due to its glycosylphosphatidylinositol (GPI) anchor, uPAR has no intracellular domain and thus exerts its signaling capacity through lateral interactions with other components of the plasma membrane that actually mediate uPAR-induced signals. As yet, a total 42 proteins interacting directly with uPAR can be numbered comprising 9 soluble ligands and 33 lateral partners. The fact that uPAR interacts with members of three major families of membrane receptors i.e. G protein-coupled receptors, receptor tyrosine kinases, and integrins implies that the actual number of components constituting the uPAR interacome is extremely high. For example, 156 factors belong to the integrin adhesome. Moreover, in the light of the wide diversity of the components of the uPAR interactome, uPAR appears to be an essential player of major biological systems including the blood coagulation, complement and plasma kallikrein-kinin cascades. This review describes the soluble ligands and lateral partners of the uPAR interactome, the mechanisms regulating uPAR interactions and their proved and/or potential biological functions.

The urokinase receptor in the central nervous system.

The urokinase receptor (uPAR) is a multifunctional glycosylphosphatidylinositol-anchored protein that regulates important processes such as gene expression, cell proliferation, adhesion, migration, invasion, and metastasis. uPAR is an essential component of the plasminogen activation cascade, a protease receptor that binds the urokinase-type plasminogen activator. uPAR is also an adhesion-modulating receptor, and a signalling receptor transmitting signals to the cell through lateral interactions with a wide array of membrane receptors. Altogether, the external ligands and membrane-bound partners of uPAR constitute a rich uPAR interactome. Recently, a new ligand of uPAR has been identified as the SRPX2 protein which is essential in language and cognitive development. SRPX2 is the second identified gene involved in language disorders. However, previous studies revealed cognitive disorders and defects in the development of the GABAergic interneurons in uPAR null mice. In addition, the expression of uPAR correlates with important human diseases such as epilepsy, autism, multiple sclerosis, Alzheimer's, AIDS dementia, cerebral malaria, and brain tumours. Therefore, uPAR has unexpectedly become a significant receptor in the central nervous system and made a few steps into philosophy. Language is indeed intimately linked to human culture. This in-depth review presents the structure and the sequences of uPAR that are essential for drug design and the generation of new inhibitors. In addition, we summarize all the inhibitors of uPAR that have been created so far. Finally, we discuss the functions of uPAR in the development, functioning, and pathology of the central nervous system.

The gammaTuRC revisited: a comparative analysis of interphase and mitotic human gammaTuRC redefines the set of core components and identifies the novel subunit GCP8.

The γ-tubulin complex is a multi-subunit protein complex that nucleates microtubule polymerization. γ-Tubulin complexes are present in all eukaryotes, but size and subunit composition vary. In Drosophila, Xenopus, and humans large γ-tubulin ring complexes (γTuRCs) have been described, which have a characteristic open ring-shaped structure and are composed of a similar set of subunits, named γ-tubulin, GCPs 2-6, and GCP-WD in humans. Despite the identification of these proteins, γTuRC function and regulation remain poorly understood. Here we establish a new method for the purification of native human γTuRC. Using mass spectrometry of whole protein mixtures we compared the composition of γTuRCs from nonsynchronized and mitotic human cells. Based on our analysis we can define core subunits as well as more transient interactors such as the augmin complex, which associates specifically with mitotic γTuRCs. We also identified GCP8/MOZART2 as a novel core subunit that is present in both interphase and mitotic γTuRCs. GCP8 depletion does not affect γTuRC assembly but interferes with γTuRC recruitment and microtubule nucleation at interphase centrosomes without disrupting general centrosome structure. GCP8-depleted cells do not display any obvious mitotic defects, suggesting that GCP8 specifically affects the organization of the interphase microtubule network.

SPICE--a previously uncharacterized protein required for centriole duplication and mitotic chromosome congression.

Proper assembly and function of a bipolar mitotic spindle is crucial for faithful bidirectional chromosome segregation during cell division. In animal cells, the two poles of the mitotic spindle are organized by centrosomes, microtubule-organizing structures composed of a pair of centrioles surrounded by the so-called pericentriolar material. Proteomic studies have revealed a large number of centrosome proteins, but many remain uncharacterized. Here, we characterize SPICE, a protein that localizes to spindle microtubules in mitosis and to centrioles throughout the cell cycle. RNAi-mediated depletion of SPICE in human cells impairs centriole duplication and causes severe mitotic defects. SPICE depletion compromises spindle architecture, spindle pole integrity and chromosome congression, even in cells in which centriole duplication has occurred. Our data suggest that SPICE is an important dual-function regulator required for centriole duplication and for proper bipolar spindle formation and chromosome congression in mitosis.

Zeolin. A new recombinant storage protein constructed using maize gamma-zein and bean phaseolin.

The major seed storage proteins of maize (Zea mays) and bean (Phaseolus vulgaris), zein and phaseolin, accumulate in the endoplasmic reticulum (ER) and in storage vacuoles, respectively. We show here that a chimeric protein composed of phaseolin and 89 amino acids of gamma-zein, including the repeated and the Pro-rich domains, maintains the main characteristics of wild-type gamma-zein: It is insoluble unless its disulfide bonds are reduced and forms ER-located protein bodies. Unlike wild-type phaseolin, the protein, which we called zeolin, accumulates to very high amounts in leaves of transgenic tobacco (Nicotiana tabacum). A relevant proportion of the ER chaperone BiP is associated with zeolin protein bodies in an ATP-sensitive fashion. Pulse-chase labeling confirms the high affinity of BiP to insoluble zeolin but indicates that, unlike structurally defective proteins that also extensively interact with BiP, zeolin is highly stable. We conclude that the gamma-zein portion is sufficient to induce the formation of protein bodies also when fused to another protein. Because the storage proteins of cereals and legumes nutritionally complement each other, zeolin can be used as a starting point to produce nutritionally balanced and highly stable chimeric storage proteins.