A MICU1 Splice Variant Confers High Sensitivity to the Mitochondrial Ca2+ Uptake Machinery of Skeletal Muscle.

Skeletal muscle is a dynamic organ, characterized by an incredible ability to rapidly increase its rate of energy consumption to sustain activity. Muscle mitochondria provide most of the ATP required for contraction via oxidative phosphorylation. Here we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro-exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca2+ one order of magnitude more efficiently than MICU1 and, when heterodimerized with MICU2, activates MCU current at lower Ca2+ concentrations than MICU1-MICU2 heterodimers. In skeletal muscle in vivo, MICU1.1 is required for sustained mitochondrial Ca2+ uptake and ATP production. These results highlight a novel mechanism of the molecular plasticity of the MCU Ca2+ uptake machinery that allows skeletal muscle mitochondria to be highly responsive to sarcoplasmic [Ca2+] responses.

A novel genetically-encoded bicyclic peptide inhibitor of human urokinase-type plasminogen activator with better cross-reactivity toward the murine orthologue.

The inhibition of human urokinase-type plasminogen activator (huPA), a serine protease that plays an important role in pericellular proteolysis, is a promising strategy to decrease the invasive and metastatic activity of tumour cells. However, the generation of selective small molecule huPA inhibitors has proven to be challenging due to the high structural similarity of huPA to other paralogue serine proteases. Efforts to generate more specific therapies have led to the development of cyclic peptide-based inhibitors with much higher selectivity against huPA. While this latter property is desired, the sparing of the orthologue murine poses difficulties for the testing of the inhibitor in preclinical mouse model. In this work, we have applied a Darwinian evolution-based approach to identify phage-encoded bicyclic peptide inhibitors of huPA with better cross-reactivity towards murine uPA (muPA). The best selected bicyclic peptide (UK132) inhibited huPA and muPA with Ki values of 0.33 and 12.58 µM, respectively. The inhibition appears to be specific for uPA, as UK132 only weakly inhibits a panel of structurally similar serine proteases. Removal or substitution of the second loop with one not evolved in vitro led to monocyclic and bicyclic peptide analogues with lower potency than UK132. Moreover, swapping of 1,3,5-tris-(bromomethyl)-benzene with different small molecules not used in the phage selection, resulted in an 80-fold reduction of potency, revealing the important structural role of the branched cyclization linker. Further substitution of an arginine in UK132 to a lysine resulted in a bicyclic peptide UK140 with enhanced inhibitory potency against both huPA (Ki = 0.20 µM) and murine orthologue (Ki = 2.79 µM). By combining good specificity, nanomolar affinity and a low molecular mass, the bicyclic peptide inhibitor developed in this work may provide a novel human and murine cross-reactive lead for the development of a potent and selective anti-metastatic therapy.

The Role of Proteolysis in Amyloidosis.

Amyloidoses are a group of diseases associated with deposits of amyloid fibrils in different tissues. So far, 36 different types of amyloidosis are known, each due to the misfolding and accumulation of a specific protein. Amyloid deposits can be found in several organs, including the heart, brain, kidneys, and spleen, and can affect single or multiple organs. Generally, amyloid-forming proteins become prone to aggregate due to genetic mutations, acquired environmental factors, excessive concentration, or post-translational modifications. Interestingly, amyloid aggregates are often composed of proteolytic fragments, derived from the degradation of precursor proteins by yet unidentified proteases, which display higher amyloidogenic tendency compared to precursor proteins, thus representing an important mechanism in the onset of amyloid-based diseases. In the present review, we summarize the current knowledge on the proteolytic susceptibility of three of the main human amyloidogenic proteins, i.e., transthyretin, ß-amyloid precursor protein, and α-synuclein, in the onset of amyloidosis. We also highlight the role that proteolytic enzymes can play in the crosstalk between intestinal inflammation and amyloid-based diseases.

Structural Features and Toxicity of α-Synuclein Oligomers Grown in the Presence of DOPAC.

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.

Sperm Cholesterol Content Modifies Sperm Function and TRPV1-Mediated Sperm Migration.

Transient receptor potential channels-vanilloid receptor 1 (TRPV1) regulates thermotaxis in sperm-oriented motility. We investigated the role of membrane cholesterol (Chol) on TRPV1-mediated human sperm migration. Semen samples were obtained from five normozoospemic healthy volunteers. Sperm membrane Chol content, quantified by liquid chromatography-mass spectrometry, was modified by incubating cells with 2-hydroxypropyl-ß-cyclodextrin (CD) or the complex between CD and Chol (CD:Chol). The effect on sperm migration on a 10 µM capsaicin gradient (CPS), a TRPV1 agonist, was then investigated. Motility parameters were evaluated by Sperm Class Analyser. Intracellular calcium concentration and acrosome reaction were measured by staining with calcium orange and FITC-conjugated anti-CD46 antibody, respectively. TRPV1-Chol interaction was modelled by computational molecular-modelling (MM). CD and CD:Chol, respectively, reduced and increased membrane Chol content in a dose-dependent manner, resulting in a dose-dependent increase and reduction of sperm migration in a CPS gradient. MM confirmed a specific interaction of Chol with a TRPV1 domain that appeared precluded to the Chol epimer epicholesterol (Epi-Chol). Accordingly, CD:Epi-Chol was significantly less efficient than CD:Chol, in reducing sperm migration under CPS gradient. Chol inhibits TRPV1-mediated sperm function by directly interacting with a consensus sequence of the receptor.

Von Willebrand Factor Multimers and the Relaxation Response: A One-Year Study.

BACKGROUND AND AIM: Mental stress represents a pivotal factor in cardiovascular diseases. The mechanism by which stress produces its deleterious ischemic effects is still under study but some of the most explored pathways are inflammation, endothelial function and balancing of the thrombotic state. In this scenario, von Willebrand factor (vWF) is a plasma glycoprotein best known for its crucial hemostatic role, also acting as key regulatory element of inflammation, being released by the activated vascular endothelium. Antistress techniques seem to be able to slow down inflammation. As we have recently verified how the practice of the Relaxation Response (RR), which counteracts psychological stress, causes favorable changes in some inflammatory genes' expressions, neurotransmitters, hormones, cytokines and inflammatory circulating microRNAs with coronary endothelial function improvement, we aimed to verify a possible change even in serum levels of vWF. Experimental procedure: We measured vWF multimers and the total protein carbonyl contents in the sera of 90 patients with ischemic heart disease (and 30 healthy controls) immediately before and after an RR session, three times (baseline, 6 months, 12 months), during a one-year follow-up study. RESULTS: According to our data, large vWF multimers decrease during the RR, as does the plasma total carbonyl content. CONCLUSION: vWF levels seem to vary rapidly between anti-inflammatory and antithrombotic behaviors dependent on psychological activity, leading to relaxation and also possibly changes in its quaternary structure.

Non-canonical proteolytic activation of human prothrombin by subtilisin from Bacillus subtilis may shift the procoagulant-anticoagulant equilibrium toward thrombosis.

Blood coagulation is a finely regulated physiological process culminating with the factor Xa (FXa)-mediated conversion of the prothrombin (ProT) zymogen to active α-thrombin (αT). In the prothrombinase complex on the platelet surface, FXa cleaves ProT at Arg-271, generating the inactive precursor prethrombin-2 (Pre2), which is further attacked at Arg-320-Ile-321 to yield mature αT. Whereas the mechanism of physiological ProT activation has been elucidated in great detail, little is known about the role of bacterial proteases, possibly released in the bloodstream during infection, in inducing blood coagulation by direct proteolytic ProT activation. This knowledge gap is particularly concerning, as bacterial infections are frequently complicated by severe coagulopathies. Here, we show that addition of subtilisin (50 nm to 2 µm), a serine protease secreted by the non-pathogenic bacterium Bacillus subtilis, induces plasma clotting by proteolytically converting ProT into active σPre2, a nicked Pre2 derivative with a single cleaved Ala-470-Asn-471 bond. Notably, we found that this non-canonical cleavage at Ala-470-Asn-471 is instrumental for the onset of catalysis in σPre2, which was, however, reduced about 100-200-fold compared with αT. Of note, σPre2 could generate fibrin clots from fibrinogen, either in solution or in blood plasma, and could aggregate human platelets, either isolated or in whole blood. Our findings demonstrate that alternative cleavage of ProT by proteases, even by those secreted by non-virulent bacteria such as B. subtilis, can shift the delicate procoagulant-anticoagulant equilibrium toward thrombosis.

Noncoded amino acids in protein engineering: Structure-activity relationship studies of hirudin-thrombin interaction.

The advent of recombinant DNA technology allowed to site-specifically insert, delete, or mutate almost any amino acid in a given protein, significantly improving our knowledge of protein structure, stability, and function. Nevertheless, a quantitative description of the physical and chemical basis that makes a polypeptide chain to efficiently fold into a stable and functionally active conformation is still elusive. This mainly originates from the fact that nature combined, in a yet unknown manner, different properties (i.e., hydrophobicity, conformational propensity, polarizability, and hydrogen bonding capability) into the 20 standard natural amino acids, thus making difficult, if not impossible, to univocally relate the change in protein stability or function to the alteration of physicochemical properties caused by amino acid exchange(s). In this view, incorporation of noncoded amino acids with tailored side chains, allowing to finely tune the structure at a protein site, would facilitate to dissect the effects of a given mutation in terms of one or a few physicochemical properties, thus much expanding the scope of physical organic chemistry in the study of proteins. In this review, relevant applications from our laboratory will be presented on the use of noncoded amino acids in structure-activity relationships studies of hirudin binding to thrombin.

Onconase dimerization through 3D domain swapping: structural investigations and increase in the apoptotic effect in cancer cells.

Onconase® (ONC), a protein extracted from the oocytes of the Rana pipiens frog, is a monomeric member of the secretory 'pancreatic-type' RNase superfamily. Interestingly, ONC is the only monomeric ribonuclease endowed with a high cytotoxic activity. In contrast with other monomeric RNases, ONC displays a high cytotoxic activity. In this work, we found that ONC spontaneously forms dimeric traces and that the dimer amount increases about four times after lyophilization from acetic acid solutions. Differently from RNase A (bovine pancreatic ribonuclease) and the bovine seminal ribonuclease, which produce N- and C-terminal domain-swapped conformers, ONC forms only one dimer, here named ONC-D. Cross-linking with divinylsulfone reveals that this dimer forms through the three-dimensional domain swapping of its N-termini, being the C-terminus blocked by a disulfide bond. Also, a homology model is proposed for ONC-D, starting from the well-known structure of RNase A N-swapped dimer and taking into account the results obtained from spectroscopic and stability analyses. Finally, we show that ONC is more cytotoxic and exerts a higher apoptotic effect in its dimeric rather than in its monomeric form, either when administered alone or when accompanied by the chemotherapeutic drug gemcitabine. These results suggest new promising implications in cancer treatment.

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.

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.

From haemadin to haemanorm: Synthesis and characterization of full-length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm).

Hirudin from Hirudo medicinalis is a bivalent α-Thrombin (αT) inhibitor, targeting the enzyme active site and exosite-I, and is currently used in anticoagulant therapy along with its simplified analogue hirulog. Haemadin, a small protein (57 amino acids) isolated from the land-living leech Haemadipsa sylvestris, selectively inhibits αT with a potency identical to that of recombinant hirudin (KI = 0.2 pM), with which it shares a common disulfide topology and overall fold. At variance with hirudin, haemadin targets exosite-II and therefore (besides the free protease) it also blocks thrombomodulin-bound αT without inhibiting the active intermediate meizothrombin, thus offering potential advantages over hirudin. Here, we produced in reasonably high yields and pharmaceutical purity (>98%) wild-type haemadin and the oxidation resistant Met5 → nor-Leucine analogue, both inhibiting αT with a KI of 0.2 pM. Thereafter, we used site-directed mutagenesis, spectroscopic, ligand-displacement, and Hydrogen/Deuterium Exchange-Mass Spectrometry techniques to map the αT regions relevant for the interaction with full-length haemadin and with the synthetic N- and C-terminal peptides Haem(1-10) and Haem(45-57). Haem(1-10) competitively binds to/inhibits αT active site (KI = 1.9 µM) and its potency was enhanced by 10-fold after Phe3 → ß-Naphthylalanine exchange. Conversely to full-length haemadin, haem(45-57) displays intrinsic affinity for exosite-I (KD = 1.6 µM). Hence, we synthesized a peptide in which the sequences 1-9 and 45-57 were joined together through a 3-Glycine spacer to yield haemanorm, a highly potent (KI = 0.8 nM) inhibitor targeting αT active site and exosite-I. Haemanorm can be regarded as a novel class of hirulog-like αT inhibitors with potential pharmacological applications.

The role of high molecular weight hyaluronic acid in mucoadhesion on an ocular surface model.

Hyaluronic acid (HA) is frequently formulated in eye drops to improve the stability of the tear film by hydration and lubrication. Mucoadhesion is related to the ocular residence time and therefore to the effectiveness of the eye drops. The ocular residence time of the HA formulation is correlated with the ability of HA to create specific strong interactions in the ocular surface with the mucus layer, mainly composed of a mixture of secreted mucins (MUC; gel forming MUC5AC and MUC2) and shed membrane-bound soluble mucins (MUC1, MUC4, and MUC16). Dry eye disease (DED) is a multifactorial pathology of the preocular tear film with possible damage to the ocular surface classified in two types: (1) aqueous-deficient dry eye and (2) evaporative dry eye, caused by a decrease in goblet cell density that reduces MUC expression and/or by meibomian gland dysfunction, that results in a drop in the lipidic fraction of the tear film. In this work, the binding affinity between HA and MUC2 has been evaluated with three complementary approaches because the secreted MUCs play a pivotal role in the viscoelastic properties of the tear film: 1. Rheological analysis, measuring the mucoadhesive index and the complex viscosity in relation to MM (Molecular Mass) and concentration; 2. Fluorescence analysis, using a fluorescent hydrophobic probe, to investigate the conformational change of MUC2 during the interaction with the HA polymer; 3. Surface plasmon resonance analysis, used to measure the affinity between MUC2 (immobilized on the surface of a sensor chip) and the HA polymers that flowed on it at the molecular level. For all these tests, the mucoadhesive performance of the natural HA linearly increases with the MM, whereas cross-linked HA and other emollient and gelling agents (formulated in artificial tears) do not show the same mucoadhesive properties (with the exception of xanthan gum). The mucoadhesive performance of high MM HA has also been confirmed in conditions that simulate the pathological condition of the tear film during DED by decreasing the MUC2 or oleic acid concentration. Physico-chemical analysis of a series of marketed artificial tears confirms the linear correlation between the MM of the HA used in the products and the mucoadhesive index measured on the ocular surface model.

Aerosolized sulfated hyaluronan derivatives prolong the survival of K18 ACE2 mice infected with a lethal dose of SARS-CoV-2.

Despite several vaccines that are currently approved for human use to control the pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent medical need for therapeutic and prophylactic options. SARS-CoV-2 binding and entry in human cells involves interactions of its spike (S) protein with several host cell surface factors, including heparan sulfate proteoglycans (HSPGs), transmembrane protease serine 2 (TMPRSS2), and angiotensin-converting enzyme 2 (ACE2). In this paper we investigated the potential of sulphated Hyaluronic Acid (sHA), a HSPG mimicking polymer, to inhibit the binding of SARS-CoV-2 S protein to human ACE2 receptor. After the assessment of different sulfation degree of sHA backbone, a series of sHA functionalized with different hydrophobic side chains were synthesized and screened. The compound showing the highest binding affinity to the viral S protein was further characterized by surface plasmon resonance (SPR) towards ACE2 and viral S protein binding domain. Selected compounds were formulated as solutions for nebulization and, after being characterized in terms of aerosolization performance and droplet size distribution, their efficacy was assessed in vivo using the K18 human (h)ACE2 transgenic mouse model of SARS-CoV-2 infection.

Exogenous human α-Synuclein acts in vitro as a mild platelet antiaggregant inhibiting α-thrombin-induced platelet activation.

α-Synuclein (αSyn) is a small disordered protein, highly conserved in vertebrates and involved in the pathogenesis of Parkinson's disease (PD). Indeed, αSyn amyloid aggregates are present in the brain of patients with PD. Although the pathogenic role of αSyn is widely accepted, the physiological function of this protein remains elusive. Beyond the central nervous system, αSyn is expressed in hematopoietic tissue and blood, where platelets are a major cellular host of αSyn. Platelets play a key role in hemostasis and are potently activated by thrombin (αT) through the cleavage of protease-activated receptors. Furthermore, both αT and αSyn could be found in the same spatial environment, i.e. the platelet membrane, as αT binds to and activates platelets that can release αSyn from α-granules and microvesicles. Here, we investigated the possibility that exogenous αSyn could interfere with platelet activation induced by different agonists in vitro. Data obtained from distinct experimental techniques (i.e. multiple electrode aggregometry, rotational thromboelastometry, immunofluorescence microscopy, surface plasmon resonance, and steady-state fluorescence spectroscopy) on whole blood and platelet-rich plasma indicate that exogenous αSyn has mild platelet antiaggregating properties in vitro, acting as a negative regulator of αT-mediated platelet activation by preferentially inhibiting P-selectin expression on platelet surface. We have also shown that both exogenous and endogenous (i.e. cytoplasmic) αSyn preferentially bind to the outer surface of activated platelets. Starting from these findings, a coherent model of the antiplatelet function of αSyn is proposed.

NOG-Derived Peptides Can Restore Neuritogenesis on a CRASH Syndrome Cell Model.

Homo- and heterophilic binding mediated by the immunoglobulin (Ig)-like repeats of cell adhesion molecules play a pivotal role in cell-cell and cell-extracellular matrix interactions. L1CAM is crucial to neuronal differentiation, in both mature and developing nervous systems, and several studies suggest that its functional interactions are mainly mediated by Ig2-Ig2 binding. X-linked mutations in the human L1CAM gene are summarized as L1 diseases, including the most diagnosed CRASH neurodevelopmental syndrome. In silico simulations provided a molecular rationale for CRASH phenotypes resulting from mutations I179S and R184Q in the homophilic binding region of Ig2. A synthetic peptide reproducing such region could both mimic the neuritogenic capacity of L1CAM and rescue neuritogenesis in a cellular model of the CRASH syndrome, where the full L1CAM ectodomain proved ineffective. Presented functional evidence opens the route to the use of L1CAM-derived peptides as biotechnological and therapeutic tools.

Preliminary Discovery of Small Molecule Inhibitors of Epidermal Growth Factor Receptor (EGFR) That Bind to the Extracellular Domain.

The Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein belonging to the protein kinase superfamily. It is composed of an extracellular domain, a transmembrane anchoring region and a cytoplasmic region endowed with tyrosine kinase activity. Genetic mutations of EGFR kinase cause higher activity thereby stimulating downstream signaling pathways that, in turn, impact transcription and cell cycle progression. Due to the involvement of mutant EGFR in tumors and inflammatory diseases, in the past decade, several EGFR inhibitory strategies have been extensively studied, either targeting the extracellular domain (through monoclonal antibodies) or the intracellular kinase domain (through ATP-mimic small molecules). Monoclonal antibodies impair the binding to growth factor, the receptor dimerization, and its activation, whereas small molecules block the intracellular catalytic activity. Herein, we describe the development of a novel small molecule, called DSF-102, that interacts with the extracellular domain of EGFR. When tested in vitro in KRAS mutant A549 cells, it impairs EGFR activity by exerting (i) dose-dependent toxicity effects; (ii) a negative regulation of ERK, MAPK p38 and AKT; and (iii) a modulation of the intracellular trafficking and lysosomal degradation of EGFR. Interestingly, DSF-102 exerts its EGFR inhibitory activity without showing interaction with the intracellular kinase domain. Taken together, these findings suggest that DSF-102 is a promising hit compound for the development of a novel class of anti-EGFR compounds, i.e., small molecules able to interact with the extracellular domain of EGFR and useful for overcoming the KRAS-driven resistance to TKI treatment.

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.

Mapping specificity, cleavage entropy, allosteric changes and substrates of blood proteases in a high-throughput screen.

Proteases are among the largest protein families and critical regulators of biochemical processes like apoptosis and blood coagulation. Knowledge of proteases has been expanded by the development of proteomic approaches, however, technology for multiplexed screening of proteases within native environments is currently lacking behind. Here we introduce a simple method to profile protease activity based on isolation of protease products from native lysates using a 96FASP filter, their analysis in a mass spectrometer and a custom data analysis pipeline. The method is significantly faster, cheaper, technically less demanding, easy to multiplex and produces accurate protease fingerprints. Using the blood cascade proteases as a case study, we obtain protease substrate profiles that can be used to map specificity, cleavage entropy and allosteric effects and to design protease probes. The data further show that protease substrate predictions enable the selection of potential physiological substrates for targeted validation in biochemical assays.

Endocrine disruption of vitamin D activity by perfluoro-octanoic acid (PFOA).

Perfluoroalkyl substances (PFAS) are a class of compounds used in industry and consumer products. Perfluorooctanoic acid (PFOA) is the predominant form in human samples and has been shown to induce severe health consequences, such as neonatal mortality, neurotoxicity, and immunotoxicity. Toxicological studies indicate that PFAS accumulate in bone tissues and cause altered bone development. Epidemiological studies have reported an inverse relationship between PFAS and bone health, however the associated mechanisms are still unexplored. Here, we present computational, in silico and in vitro evidence supporting the interference of PFOA on vitamin D (VD). First, PFOA competes with calcitriol on the same binding site of the VD receptor, leading to an alteration of the structural flexibility and a 10% reduction by surface plasmon resonance analysis. Second, this interference leads to an altered response of VD-responsive genes in two cellular targets of this hormone, osteoblasts and epithelial cells of the colorectal tract. Third, mineralization in human osteoblasts is reduced upon coincubation of PFOA with VD. Finally, in a small cohort of young healthy men, PTH levels were higher in the exposed group, but VD levels were comparable. Altogether these results provide the first evidence of endocrine disruption by PFOA on VD pathway by competition on its receptor and subsequent inhibition of VD-responsive genes in target cells.