In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A.

In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.

Halogen-Mediated Partial Oxidation of Polyvinyl Alcohol for Tissue Engineering Purposes.

Partial oxidation of polyvinyl alcohol (PVA) with potassium permanganate turned out to be an efficient method to fabricate smart scaffolds for tissue engineering, endowed with biodegradation and protein delivery capacity. This work considered for the first time the use of halogens (bromine, chlorine and iodine) as less aggressive agents than potassium permanganate to perform controlled PVA oxidation, in order to prevent degradation of polymer molecular size upon chemical modification. Oxidized PVA solutions were chemically characterized (i.e., dinitrophenylhydrazine assay, viscosity measurements, molecular size distribution) before preparing physically cross-linked hydrogels. Scaffolds were assessed for their mechanical properties and cell/tissue biocompatibiliy through cytotoxic extract test on IMR-90 fibroblasts and subcutaneous implantation into BALB/c mice. According to chemical investigations, bromine and iodine allowed for minor alteration of polymer molecular weight. Uniaxial tensile tests demonstrated that oxidized scaffolds had decreased mechanical resistance to deformation, suggesting tunable hydrogel stiffness. Finally, oxidized hydrogels exhibited high biocompatibility both in vitro and in vivo, resulting neither to be cytotoxic nor to elicit severe immunitary host reaction in comparison with atoxic PVA. In conclusion, PVA hydrogels oxidized by halogens were successfully fabricated in the effort of adapting polymer characteristics to specific tissue engineering applications.

New bioresorbable wraps based on oxidized polyvinyl alcohol and leukocyte-fibrin-platelet membrane to support peripheral nerve neurorrhaphy: preclinical comparison versus NeuraWrap.

Nerve wrapping improves neurorrhaphy outcomes in case of peripheral nerve injuries (PNIs). The aim of this preclinical study was to assess the efficacy of two novel biodegradable wraps made of a synthetic 1% oxidized polyvinyl alcohol (OxPVA) and a natural leukocyte-fibrin-platelet membrane (LFPm) versus the commercial product NeuraWrap. After rats sciatic nerve transection and neurorrhaphy, the wraps were implanted and compared for functional outcome, by sciatic function index assessment; structural characteristics, by histological/immunohistochemical analysis; ultrastructural features, by transmission electron microscopy. Moreover, a morphometric study was also performed and collagen distribution was observed by Second Harmonic Generation microscopy. After 12 weeks from implantation, all wraps assured nerve function recovery; no scar tissue/neuromas were visible at dissection. LFPm wraps were completely resorbed, while residues of OxPVA and NeuraWrap were observed. In all groups, biocompatibility was confirmed by the absence of significant inflammatory infiltrate. According to histological/immunohistochemical analysis and morphometric findings, OxPVA and LFPm wraps were both effective in preserving nerve integrity. These results assess that bioengineered OxPVA and LFPm wraps successfully guarantee favorable lesion recovery after PNI/neurorrhaphy and, in future, may be considered an interesting alternative to the commercial NeuraWrap.

Development of Oxidized Polyvinyl Alcohol-Based Nerve Conduits Coupled with the Ciliary Neurotrophic Factor.

Functionalized synthetic conduits represent a promising strategy to enhance peripheral nerve regeneration by guiding axon growth while delivering therapeutic neurotrophic factors. In this work, hollow nerve conduits made of polyvinyl alcohol partially oxidized with bromine (OxPVA_Br2) and potassium permanganate (OxPVA_KMnO4) were investigated for their structural/biological properties and ability to absorb/release the ciliary neurotrophic factor (CNTF). Chemical oxidation enhanced water uptake capacity of the polymer, with maximum swelling index of 60.5% ± 2.5%, 71.3% ± 3.6% and 19.5% ± 4.0% for OxPVA_Br2, OxPVA_KMnO4 and PVA, respectively. Accordingly, hydrogel porosity increased from 15.27% ± 1.16% (PVA) to 62.71% ± 8.63% (OxPVA_Br2) or 77.50% ± 3.39% (OxPVA_KMnO4) after oxidation. Besides proving that oxidized PVA conduits exhibited mechanical resistance and a suture holding ability, they did not exert a cytotoxic effect on SH-SY5Y and Schwann cells and biodegraded over time when subjected to enzymatic digestion, functionalization with CNTF was performed. Interestingly, higher amounts of neurotrophic factor were detected in the lumen of OxPVA_Br2 (0.22 ± 0.029 µg) and OxPVA_KMnO4 (0.29 ± 0.033 µg) guides rather than PVA (0.11 ± 0.021 µg) tubular scaffolds. In conclusion, we defined a promising technology to obtain drug delivery conduits based on functionalizable oxidized PVA hydrogels.

Biofabrication of a novel leukocyte-fibrin-platelet membrane as a cells and growth factors delivery platform for tissue engineering applications.

Autologous platelet-rich hemocomponents have emerged as potential biologic tools for regenerative purpose, but their therapeutic efficacy still remains controversial. This work represents the characterization study of an innovative autologous leukocyte-fibrin-platelet membrane (LFPm), which we prepared according to a novel protocol involving multiple cycles of apheresis. The high content in fibrinogen gave to our hemocomponent the appearance of a manipulable and suturable membrane with high elasticity and deformation capacity. Moreover, being highly enriched with platelets, leukocytes, and monocytes/macrophages, the LFPm sustained the local release of bioactive molecules (platelet derived growth factor, vascular endothelial growth factor, interleukin-10, and tumour necrosis factor alpha). In parallel, the evaluation of stemness potential highlighted also that the LFPm contained cells expressing pluripotency and multipotency markers both at the messenger ribonucleic acid (NANOG, SOX2, THY1, NT5E, and ENG) and surface-protein level (CD44high /CD73+ /CD34+ /CD117+ /CD31+ ). Finally, biodegradation analysis interestingly showed a good stability of the membrane for at least 3 weeks in vitro and 1 week in vivo. In both cases, biodegradation was associated with progressive exposure of fibrin scaffold, loss/migration of cellular elements, and release of growth factors. Overall, collected evidence could shed some light on the regenerative effect that LFPms may exert after the autologous implant on a defect site.

Partially oxidized polyvinyl alcohol conduitfor peripheral nerve regeneration.

Surgical reconstruction of peripheral nerves injuries with wide substance-loss is still a challenge. Many studies focused on the development of artificial nerve conduits made of synthetic or biological materials but the ideal device has not yet been identified. Here, we manufactured a conduit for peripheral nerve regeneration using a novel biodegradable hydrogel we patented that is oxidized polyvinyl alcohol (OxPVA). Thus, its characteristics were compared with neat polyvinyl alcohol (PVA) and silk-fibroin (SF) conduits, through in vitro and in vivo analysis. Unlike SF, OxPVA and neat PVA scaffolds did not support SH-SY5Y adhesion and proliferation in vitro. After implantation in rat model of sciatic nerve transection, the three conduits sustained the regeneration of the injured nerve filling a gap of 5 mm in 12 weeks. Implanted animals showed a good gait recovery. Morphometric data related to the central portion of the explanted conduit interestingly highlighted a significantly better outcome for OxPVA scaffolds compared to PVA conduits in terms of axon density, also with respect to the autograft group. This study suggests the potential of our novel biomaterial for the development of conduits for clinical use in case of peripheral nerve lesions with substance loss.

Partially oxidized polyvinyl alcohol as a promising material for tissue engineering.

The desired clinical outcome after implantation of engineered tissue substitutes depends strictly on the development of biodegradable scaffolds. In this study we fabricated 1% and 2% oxidized polyvinyl alcohol (PVA) hydrogels, which were considered for the first time for tissue-engineering applications. The final aim was to promote the protein release capacity and biodegradation rate of the resulting scaffolds in comparison with neat PVA. After physical crosslinking, characterization of specific properties of 1% and 2% oxidized PVA was performed. We demonstrated that mechanical properties, hydrodynamic radius of molecules, thermal characteristics and degree of crystallinity were inversely proportional to the PVA oxidation rate. On the other hand, swelling behaviour and protein release were enhanced, confirming the potential of oxidized PVA as a protein delivery system, besides being highly biodegradable. Twelve weeks after in vivo implantation in mice, the modified hydrogels did not elicit severe inflammatory reactions, showing them to be biocompatible and to degrade faster as the degree of oxidation increased. According to our results, oxidized PVA stands out as a novel biomaterial for tissue engineering that can be used to realize scaffolds with customizable mechanical behaviour, protein-loading ability and biodegradability. Copyright © 2015 John Wiley & Sons, Ltd.

In vitro assessment of TAT - Ciliary Neurotrophic Factor therapeutic potential for peripheral nerve regeneration.

In regenerative neurobiology, Ciliary Neurotrophic Factor (CNTF) is raising high interest as a multifunctional neurocytokine, playing a key role in the regeneration of injured peripheral nerves. Despite its promising trophic and regulatory activity, its clinical application is limited by the onset of severe side effects, due to the lack of efficient intracellular trafficking after administration. In this study, recombinant CNTF linked to the transactivator transduction domain (TAT) was investigated in vitro and found to be an optimized fusion protein which preserves neurotrophic activity, besides enhancing cellular uptake for therapeutic advantage. Moreover, a compelling protein delivery method was defined, in the future perspective of improving nerve regeneration strategies. Following determination of TAT-CNTF molecular weight and concentration, its specific effect on neural SH-SY5Y and PC12 cultures was assessed. Cell proliferation assay demonstrated that the fusion protein triggers PC12 cell growth within 6h of stimulation. At the same time, the activation of signal transduction pathway and enhancement of cellular trafficking were found to be accomplished in both neural cell lines after specific treatment with TAT-CNTF. Finally, the recombinant growth factor was successfully loaded on oxidized polyvinyl alcohol (PVA) scaffolds, and more efficiently released when polymer oxidation rate increased. Taken together, our results highlight that the TAT domain addiction to the protein sequence preserves CNTF specific neurotrophic activity in vitro, besides improving cellular uptake. Moreover, oxidized PVA could represent an ideal biomaterial for the development of nerve conduits loaded with the fusion protein to be delivered to the site of nerve injury.

Autologous chondrocytes as a novel source for neo-chondrogenesis in haemophiliacs.

Haemophilic arthropathy is the major cause of disability in patients with haemophilia and, despite prophylaxis with coagulation factor concentrates, some patients still develop articular complications. We evaluate the feasibility of a tissue engineering approach to improve current clinical strategies for cartilage regeneration in haemophiliacs by using autologous chondrocytes (haemophilic chondrocytes; HaeCs). Little is known about articular chondrocytes from haemophilic patients and no characterisation has as yet been performed. An investigation into whether blood exposure alters HaeCs should be interesting from the perspective of autologous implants. The typical morphology and expression of specific target genes and surface markers were therefore assessed by optical microscopy, reverse transcription plus the polymerase chain reaction (PCR), real-time PCR and flow-cytometry. We then considered chondrocyte behaviour on a bio-hybrid scaffold (based on polyvinyl alcohol/Wharton's jelly) as an in vitro model of articular cartilage prosthesis. Articular chondrocytes from non-haemophilic donors were used as controls. HaeC morphology and the resulting immunophenotype CD44(+)/CD49c(+)/CD49e(+)/CD151(+)/CD73(+)/CD49f(-)/CD26(-) resembled those of healthy donors. Moreover, HaeCs were active in the transcription of genes involved in the synthesis of the extracellular matrix proteins of the articular cartilage (ACAN, COL1A, COL2A, COL10A, COL9A, COMP, HAS1, SOX9), although the over-expression of COL1A1, COL10A1, COMP and HAS was observed. In parallel, the composite scaffold showed adequate mechanical and biological properties for cartilage tissue engineering, promoting chondrocyte proliferation. Our preliminary evidence contributes to the characterisation of HaeCs, highlighting the opportunity of using them for autologous cartilage implants in patients with haemophilia.

Adrenomedullin in the growth modulation and differentiation of acute myeloid leukemia cells.

Adrenomedullin (ADM) is a regulatory peptide endowed with multiple biological effects, including the regulation of blood pressure, cell growth and innate host defence. In the present study, we demonstrated that ADM signaling could be involved in the impaired cellular differentiation of myeloid leukemia cells to mature granulocytes or monocytes by modulating RAMPs/CRLR expression, PI3K/Akt cascade and the ERK/MAPK signaling pathway. When exogenously administered to in vitro cultures of HL60 promyelocytic leukemia cells, ADM was shown to exert a strong proliferative effect with minimal upregulation in the expression level of monocyte antigen CD14. Notably, the experimental inhibition of ADM signaling with inhibitor ADM22-52 promoted a differentiative stimulation towards monocytic and granulocytic lineages. Moreover, based on the expression of CD31 relative to CD38, we hypothesized that an excess of ADM in bone marrow (BM) niche could increase the transendothelial migration of leukemia cells while any inhibitory event of ADM activity could raise cell retention in hyaluronate matrix by upregulating CD38. Taken into consideration the above evidence, we concluded that ADM and ADM22-52 could differently affect the growth of leukemia cells by autocrine/paracrine mechanisms and may have clinical relevance as biological targets for the intervention of tumor progression.

Tailored PVA/ECM scaffolds for cartilage regeneration.

Articular cartilage lesions are a particular challenge for regenerative medicine due to cartilage low self-ability repair in case of damage. Hence, a significant goal of musculoskeletal tissue engineering is the development of suitable structures in virtue of their matrix composition and biomechanical properties. The objective of our study was to design in vitro a supporting structure for autologous chondrocyte growth. We realized a biohybrid composite scaffold combining a novel and nonspecific extracellular matrix (ECM), which is decellularized Wharton's jelly ECM, with the biomechanical properties of the synthetic hydrogel polyvinyl alcohol (PVA). Wharton's jelly ECM was tested for its ability in promoting scaffold colonization by chondrocytes and compared with polyvinyl alcohol itself and the more specific decellularized cartilage matrix. Our preliminary evidences highlighted the chance of using Wharton's jelly ECM in combination with PVA hydrogels as an innovative and easily available scaffold for cartilage restoration.

Evaluation of vascular grafts based on polyvinyl alcohol cryogels.

The present study designed and developed blood vessel substitutes (BVSs) composed of polyvinyl alcohol (PVA) cryogels. The in vitro results demonstrated that the coating of the polymer with lyophilized decellularized vascular matrix (DVM) greatly enhanced the adhesion of human umbilical vein endothelial cells (HUVECs). However, when PVA̸DVM BVSs were implanted into the abdominal aorta of Sprague­Dawley rats, DVM was identified as a highly thrombogenic surface resulting in the mortality of all animals 3­4 days after surgery. By contrast, all rats implanted with PVA survived and were sacrificed after 12 months. The luminal surface of the explanted grafts was completely covered by endothelial cells and the inner diameter was similar to that of the original vessel. In conclusion, the present study indicated that PVA may be considered as a promising biomaterial for the fabrication of artificial vessels.

Assessment of in vitro temporal corrosion and cytotoxicity of AZ91D alloy.

Magnesium alloys represent a valuable option for the production of bioresorbable implantable medical devices aimed to improve the therapeutic approach and minimize the potential risks related to biostable materials. In this regard, the degradation process needs to be carefully evaluated in order to assess the effectiveness of the regenerative support and the eventual toxic effects induced by the released corrosion products. Aluminium is one of the most common alloying element that raised several safety concerns, contributing to shift the investigation toward Al-free alloys. To delve into this issue, a long-term investigation (up to 28 days) was performed using AZ91D alloy, due to its relevant Al content. Immersion tests in phosphate buffered saline (PBS) solution was performed following the ASTM standards and the corrosion behaviour was evaluated at fixed time points by means of electrochemical techniques. Cytotoxic effects were assessed by culturing human neuroblastoma cells with conditioned medium derived from immersion tests at different dilution degree. An increase in the resistance corrosion with the time was observed. In all the investigated cases the presence of Al in the conditioned media did not induce significant toxic effects directly correlated to its content. A decrease of cell viability was only observed in the case of 50 % dilution of PBS conditioned for the longest immersion period (i.e., 28 days).

ECM-based triple layered scaffolds for vascular tissue engineering.

The present study focused on the development of three layered small-diameter (<6 mm) extracellular matrix (ECM)-based vessels. These were engineered artificially through the freeze-drying technique. A layer of decellularized bovine aorta (DAM) was deposited on a mandrel and, after lyophilization, it was dipped into a poly-L-lactide acid (PLLA)/polyethylene glycol (PEG) 2000 dichloromethane solution then quickly wrapped with a pre-prepared thin DAM sheet. Mechanical properties of three-layered scaffolds were evaluated by means of uniaxial tensile measurement. Furthermore, human endothelial and smooth muscle cells were seeded on internal and external scaffold surfaces, respectively, and co-cultured for 7 days. Our results demonstrate that i) ECM components provide suitable stimuli for cell adhesion and proliferation, ii) the microporous intermediate PLLA/PEG2000 layer is responsible for the scaffold resistance and iii) the layered deposition technique can be considered a valuable method to obtain layered vascular scaffolds of different sizes and with a good compromise between stiffness and elasticity for optimal cell organization.

Decellularized bovine reinforced vessels for small-diameter tissue-engineered vascular grafts.

The aim of the present study was to investigate the influence of a decellularization protocol on the structure and the mechanical behavior of small-diameter (<6 mm) tibial calf arteries and veins. Calf vessels were decellularized by a detergent-enzymatic method (DEM), partially hydrolyzed with trypsin and subsequently cross-linked using poly(ethylene glycol) diglycidyl ether. Our results showed that i) the DEM can be considered a simple and valuable procedure for the preparation of complete acellular arteries and veins able to preserve a high degree of collagen and elastic fibers, and ii) poly(ethylene glycol) diglycidyl ether cross-linking treatment provides appropriate mechanical reinforcement of blood vessels. Histologically, the decellularized vessels were obtained employing the detergent-enzymatic procedure and their native extracellular matrix histoarchitecture and components remained well preserved. Moreover, the decellularization protocol can be considered an effective method to remove HLA class I antigen expression from small-diameter tibial calf arteries and veins. Cytocompatibility of decellularized cross-linked vessels was evaluated by endothelial and smooth muscle cell seeding on luminal and adventitial vessel surfaces, respectively.

Porous alginate/poly(ε-caprolactone) scaffolds: preparation, characterization and in vitro biological activity.

In bone tissue engineering, scaffolds with controlled porosity are required to allow cell ingrowth, nutrient diffusion and sufficient formation of vascular networks. The physical properties of synthetic scaffolds are known to be dependent on the biomaterial type and its processing technique. In this study, we demonstrate that the separation phase technique is a useful method to process poly(ε-caprolactone) (PCL) into a desired shape and size. Moreover, using poly(ethylene glycol), sucrose, fructose and Ca2+ alginate as porogen agents, we obtained PCL scaffolds with three-dimensional porous structures characterized by different pore size and geometry. Scanning electron microscopy and porosity analysis indicated that PCL scaffolds prepared with Ca2+ alginate threads resemble the porosity and the homogeneous pore size distribution of native bone. In parallel, MicroCT analysis confirmed the presence of interconnected void spaces suitable to guarantee a biological environment for cellular growth, as demonstrated by a biocompatibility test with MC3T3-E1 murine preosteoblastic cells. In particular, scaffolds prepared with Ca2+ alginate threads increased adhesion and proliferation of MC3T3-E1 cells under basal culture conditions, and upon stimulation with a specific differentiation culture medium they enhanced the early and later differentiated cell functions, including alkaline phosphatase activity and mineralized extracellular matrix production. These results suggest that PCL scaffolds, obtained by separation phase technique and prepared with alginate threads, could be considered as candidates for bone tissue engineering applications, possessing the required physical and biological properties.

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences.

It is well known that tumor growth is strictly dependent on neo-vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)(4)K] or the heparin-binding sequence of human vitronectin that interacts with HSPGs [HVP(351-359)]. Cell adhesion, proliferation, migration, and capillary-like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)(4)K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti-angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro-angiogenic effects induced by the Fibroblast growth factor (FGF-2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)(4)K. Our data indicate that the activity of RGD-containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti-angiogenic properties of (GRGDSP)(4)K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF-2.

Involvement of the receptor for advanced glycation-end products (RAGE) in beta-amyloid-induced toxic effects in rat cerebromicrovascular endothelial cells cultured in vitro.

To ascertain whether the potential biological effects of beta amyloid (betaA) on the endothelium are partly mediated by the receptor for advanced glycation-end products (RAGE), we performed a series of experiments which analyzed the effects of the betaA(1-42) peptide on in vitro cerebromicrovascular endothelial cells (CECs). Our results suggest that RAGE is directly responsible for betaA(1-42) actions on CECs, such as its toxic effect on cell survival, viability and angiogenic capability. We observed that a 6-h incubation period exposing CECs to betaA(1-42) increased the extracellular levels of nitrite. Furthermore, the presence of a nitric oxide synthase inhibitor, L-NAME, was able to enhance CEC survival and viability. Immunocytochemical analyses demonstrated that the peptide induced expression of the inducible form of NOS, iNOS, typically synthesized in response to immune/inflammatory stimuli. Upon blocking the interaction of betaA(1-42) and RAGE, we observed significantly decreased levels of NO and suppression of iNOS immunoreactivity. In conclusion, our data suggest the involvement of RAGE, at least partly, in mediating the effects of betaA(1-42) on CECs. In particular, the decrease of in vitro cell viability and functionality and nitrosative stress activation was inhibited by blocking betaA(1-42)-RAGE interaction.

Covalent surface modification of titanium oxide with different adhesive peptides: surface characterization and osteoblast-like cell adhesion.

A fundamental goal in the field of implantology is the design of innovative devices suitable for promoting implant-to-tissue integration. This result can be achieved by means of surface modifications aimed at optimizing tissue regeneration. In the framework of oral and orthopedic implantology, surface modifications concern both the optimization of titanium/titanium alloy surface roughness and the attachment of biochemical factors able to guide cellular adhesion and/or growth. This article focuses on the covalent attachment of two different adhesive peptides to rough titanium disks. The capability of biomimetic surfaces to increase osteoblast adhesion and the specificity of their biological activity due to the presence of cell adhesion signal-motif have also been investigated. In addition, surface analyses by profilometry, X-ray photoelectron spectroscopy, and time of flight-secondary ion mass spectrometry have been carried out to investigate the effects and modifications induced by grafting procedures.

o-Nitrotyrosine and p-iodophenylalanine as spectroscopic probes for structural characterization of SH3 complexes.

High-throughput screening of protein-protein and protein-peptide interactions is of high interest both for biotechnological and pharmacological applications. Here, we propose the use of the noncoded amino acids o-nitrotyrosine and p-iodophenylalanine as spectroscopic probes in combination with circular dichroism and fluorescence quenching techniques (i.e., collisional quenching and resonance energy transfer) as a means to determine the peptide orientation in complexes with SH3 domains. Proline-rich peptides bind SH3 modules in two alternative orientations, according to their sequence motifs, classified as class I and class II. The method was tested on an SH3 domain from a yeast myosin that is known to recognize specifically class I peptides. We exploited the fluorescence quenching effects induced by o-nitrotyrosine and p-iodophenylalanine on the fluorescence signal of a highly conserved Trp residue, which is the signature of SH3 domains and sits directly in the binding pocket. In particular, we studied how the introduction of the two probes at different positions of the peptide sequence (i.e., N-terminally or C-terminally) influences the spectroscopic properties of the complex. This approach provides clear-cut evidence of the orientation of the binding peptide in the SH3 pocket. The chemical strategy outlined here can be easily extended to other protein modules, known to bind linear sequence motifs in a highly directional manner.