A conserved Neurite Outgrowth and Guidance motif with biomimetic potential in neuronal Cell Adhesion Molecules.

The discovery of conserved protein motifs can, in turn, unveil important regulatory signals, and when properly designed, synthetic peptides derived from such motifs can be used as biomimetics for biotechnological and therapeutic purposes. We report here that specific Ig-like repeats from the extracellular domains of neuronal Cell Adhesion Molecules share a highly conserved Neurite Outgrowth and Guidance (NOG) motif, which mediates homo- and heterophilic interactions crucial in neural development and repair. Synthetic peptides derived from the NOG motif of such proteins can boost neuritogenesis, and this potential is also retained by peptides with recombinant sequences, when fitting the NOG sequence pattern. The NOG motif discovery not only provides one more tile to the complex puzzle of neuritogenesis, but also opens the route to new neural regeneration strategies via a tunable biomimetic toolbox.

Endocrine Disruption of Androgenic Activity by Perfluoroalkyl Substances: Clinical and Experimental Evidence.

BACKGROUND: Considerable attention has been paid to perfluoroalkyl compounds (PFCs) because of their worldwide presence in humans, wildlife, and environment. A wide variety of toxicological effects is well supported in animals, including testicular toxicity and male infertility. For these reasons, the understanding of epidemiological associations and of the molecular mechanisms involved in the endocrine-disrupting properties of PFCs on human reproductive health is a major concern. OBJECTIVE: To investigate the relationship between PFC exposure and male reproductive health. DESIGN: This study was performed within a screening protocol to evaluate male reproductive health in high schools. PATIENTS: This is a cross-sectional study on 212 exposed males from the Veneto region, one of the four areas worldwide heavily polluted with PFCs, and 171 nonexposed controls. MAIN OUTCOME MEASURES: Anthropometrics, seminal parameters, and sex hormones were measured in young males from exposed areas compared with age-matched controls. We also performed biochemical studies in established experimental models. RESULTS: We found that increased levels of PFCs in plasma and seminal fluid positively correlate with circulating testosterone (T) and with a reduction of semen quality, testicular volume, penile length, and anogenital distance. Experimental evidence points toward an antagonistic action of perfluorooctanoic acid on the binding of T to androgen receptor (AR) in a gene reporter assay, a competition assay on an AR-coated surface plasmon resonance chip, and an AR nuclear translocation assay. DISCUSSION: This study documents that PFCs have a substantial impact on human health as they interfere with hormonal pathways, potentially leading to male infertility.

Molecular mapping of α-thrombin (αT)/ß2-glycoprotein I (ß2GpI) interaction reveals how ß2GpI affects αT functions.

ß2-Glycoprotein I (ß2GpI) is the major autoantigen in the antiphospholipid syndrome, a thrombotic autoimmune disease. Nonetheless, the physiological role of ß2GpI is still unclear. In a recent work, we have shown that ß2GpI selectively inhibits the procoagulant functions of human α-thrombin (αT; i.e. prolongs fibrin clotting time, tc, and inhibits αT-induced platelet aggregation) without affecting the unique anticoagulant activity of the protease, i.e. the proteolytic generation of the anticoagulant protein C (PC) from the PC zymogen, which interacts with αT exclusively at the protease catalytic site. Here, we used several different biochemical/biophysical techniques and molecular probes for mapping the binding sites in the αT-ß2GpI complex. Our results indicate that αT exploits the highly electropositive exosite-II, which is also responsible for anchoring αT on the platelet GpIbα (platelet receptor glycoprotein Ibα) receptor, for binding to a continuous negative region on ß2GpI structure, spanning domain IV and (part of) domain V, whereas the protease active site and exosite-I (i.e. the fibrinogen-binding site) remain accessible for substrate/ligand binding. Furthermore, we provided evidence that the apparent increase in tc, previously observed with ß2GpI, is more likely caused by alteration in the ensuing fibrin structure rather than by the inhibition of fibrinogen hydrolysis. Finally, we produced a theoretical docking model of αT-ß2GpI interaction, which was in agreement with the experimental results. Altogether, these findings help to understand how ß2GpI affects αT interactions and suggest that ß2GpI may function as a scavenger of αT for binding to the GpIbα receptor, thus impairing platelet aggregation while enabling normal cleavage of fibrinogen and PC.

Osteocalcin and Sex Hormone Binding Globulin Compete on a Specific Binding Site of GPRC6A.

The undercarboxylated form of osteocalcin (ucOC) regulates male fertility and energy metabolism, acting through the G protein-coupled receptor (GPRC)6A, thus forming a new pancreas-bone-testis axis. Recently, GPRC6A has also been suggested to mediate the nongenomic responses of free testosterone (T). However, these data did not consider the physiological scenario, where circulating T is mainly bound to sex hormone-binding globulin (SHBG) and only a small percentage circulates freely in the blood. Here, by the use of computational modelling, we document the existence of similar structural moieties between ucOC and SHBG that are predicted to bind to GPRC6A at docking analysis. This hypothesis of competition was assessed by binding experiments on human embryonic kidney-293 cells transfected with human GPRC6A gene. Unliganded SHBG specifically bound the membrane of human embryonic kidney-293 cells transfected with GPRC6A and was displaced by ucOC when coincubated at 100-fold molar excess. Furthermore, specific downstream Erk1/2 phosphorylation after stimulation of GPRC6A with ucOC was significantly blunted by 100-fold molar excess of unliganded SHBG. Intriguingly previous incubation with unliganded SHBG, followed by incubation with T, induced Erk1/2 phosphorylation in a dose-dependent manner. Neither binding nor stimulating activities were shown for SHBG saturated with T. Experiments on mutation constructs of GPRC6A strengthened the hypothesis of a common binding site of ucOC and SHBG. Given the role of GPRC6A on energy metabolism, these data agree with epidemiological association between SHBG levels and insulin sensitivity, suggest GPRC6A as a likely SHBG receptor, and add bases for the possible regulation of androgen activity in a nonsteroidal manner.

Neuronal commitment of human circulating multipotent cells by carbon nanotube-polymer scaffolds and biomimetic peptides.

AIM: We aimed to set up a self-standing, biomimetic scaffold system able to induce and support per se neuronal differentiation of autologous multipotent cells. MATERIALS & METHODS: We isolated a population of human circulating multipotent cells (hCMCs), and used carbon nanotube/polymer nanocomposite scaffolds to mimic electrical/nanotopographical features of the neural environment, and biomimetic peptides reproducing axon guidance cues from neural proteins. RESULTS: hCMCs showed high degree of stemness and multidifferentiative potential; stimuli from the scaffolds and biomimetic peptides could induce and boost hCMC differentiation toward neuronal lineage despite the absence of exogenously added, specific growth factors. CONCLUSION: This work suggests the scaffold-peptides system combined with autologous hCMCs as a functional biomimetic, self-standing prototype for neural regenerative medicine applications.

Loop Electrostatics Asymmetry Modulates the Preexisting Conformational Equilibrium in Thrombin.

Thrombin exists as an ensemble of active (E) and inactive (E*) conformations that differ in their accessibility to the active site. Here we show that redistribution of the E*-E equilibrium can be achieved by perturbing the electrostatic properties of the enzyme. Removal of the negative charge of the catalytic Asp102 or Asp189 in the primary specificity site destabilizes the E form and causes a shift in the 215-217 segment that compromises substrate entrance. Solution studies and existing structures of D102N document stabilization of the E* form. A new high-resolution structure of D189A also reveals the mutant in the collapsed E* form. These findings establish a new paradigm for the control of the E*-E equilibrium in the trypsin fold.

Enhanced neuronal cell differentiation combining biomimetic peptides and a carbon nanotube-polymer scaffold.

Carbon nanotubes are attractive candidates for the development of scaffolds able to support neuronal growth and differentiation thanks to their ability to conduct electrical stimuli, to interface with cells and to mimic the neural environment. We developed a biocompatible composite scaffold, consisting of multi-walled carbon nanotubes dispersed in a poly-L-lactic acid matrix able to support growth and differentiation of human neuronal cells. Moreover, to mimic guidance cues from the neural environment, we also designed synthetic peptides, derived from L1 and LINGO1 proteins. Such peptides could positively modulate neuronal differentiation, which is synergistically improved by the combination of the nanocomposite scaffold and the peptides, thus suggesting a prototype for the development of implants for long-term neuronal growth and differentiation. From the clinical editor: The study describes the design and preparation of nanocomposite scaffolds with multi-walled carbon nanotubes in a poly-L-lactic acid matrix. This compound used in combination with peptides leads to synergistic effects in supporting neuronal cell growth and differentiation.

Uncarboxylated osteocalcin stimulates 25-hydroxy vitamin D production in Leydig cell line through a GPRC6a-dependent pathway.

Recent studies disclosed a cross talk between testis and bone. By the action of LH, Leydig cells are able to modulate bone metabolism through testosterone and insulin-like factor 3. Moreover, LH modulates the Leydig expression of CYP2R1, the key enzyme involved in vitamin D (Vit D) 25-hydroxylation. However, pathways regulating CYP2R1 expression have been poorly investigated. The cross talk from the bone to the testis of the vitamin D 25-hydroxylase CYP2R1 involves osteocalcin (OC), which is produced by the osteoblasts and stimulates the production of testosterone by the Leydig cells through its putative receptor GPRC6A, a cation-sensing G-protein-coupled receptor. The aim of this study was to investigate the possible action of OC on CYP2R1 expression and 25-hydroxy Vit D (25-OH Vit D) production in a mouse Leydig cell line (MA-10). After confirmation of the expression of GPRC6A by MA-10, we found that stimulation with either human chorionic gonadotropin or uncarboxylated-OC (ucOC) increases CYP2R1 protein expression in a dose-dependent manner and, in turn, increases the release of 25-OH Vit D in culture medium. This effect was abolished by receptor blockade with, respectively, anti-LH receptor and anti-GPRC6A antibodies. Moreover, both agonists converged to phosphorylation of Erk1/2 by a likely differential action on second messengers. Human chorionic gonadotropin induced slow "tonic" increase of intercellular calcium and accumulation of cAMP, whereas ucOC mainly induced phasic increase of cell calcium. Supporting these findings, we found that serum ucOC positively correlated with 25-OH Vit D levels in 40 overweight male patients and 21 controls. Altogether, our results suggest that OC contributes with LH to 25-OH Vit D production by Leydig cells.