Uncovering the Role of Natural and Synthetic Small Molecules in Counteracting the Burden of α-Synuclein Aggregates and Related Toxicity in Different Models of Parkinson's Disease.

A proteostasis network represents a sophisticated cellular system that controls the whole process which leads to properly folded functional proteins. The imbalance of proteostasis determines a quantitative increase in misfolded proteins prone to aggregation and elicits the onset of different diseases. Among these, Parkinson's Disease (PD) is a progressive brain disorder characterized by motor and non-motor signs. In PD pathogenesis, alpha-Synuclein (α-Syn) loses its native structure, triggering a polymerization cascade that leads to the formation of toxic inclusions, the PD hallmark. Because molecular chaperones represent a "cellular arsenal" to counteract protein misfolding and aggregation, the modulation of their expression represents a compelling PD therapeutic strategy. This review will discuss evidence concerning the effects of natural and synthetic small molecules in counteracting α-Syn aggregation process and related toxicity, in different in vitro and in vivo PD models. Firstly, the role of small molecules that modulate the function(s) of chaperones will be highlighted. Then, attention will be paid to small molecules that interfere with different steps of the protein-aggregation process. This overview would stimulate in-depth research on already-known small molecules or the development of new ones, with the aim of developing drugs that are able to modify the progression of the disease.

Notational Analysis of Wheelchair Paralympic Table Tennis Matches.

Paralympic table tennis is the third largest paralympic sport for the number of players. Performance analysis was conducted for the rally duration and interval and impact of serve, whilst none investigated the shots distribution among classes of physical impairment. Therefore, the purpose of this study was to conduct a notational analysis of international competitions in relation to the wheelchair classes. Five matches for each wheelchair class (C1-to-C5) were evaluated from 20 elite male right-handed players. Both players for each match were analyzed for the following performance indicators: strokes type, the area of ball bouncing, and the shots outcome. Backhand shots were the most used technique for all classes. The most used strokes for C1 players were backhand and forehand drive and backhand lob, while for C5 players they were backhand and forehand push and backhand topspin. Similar shots distribution was registered for C2-to-C5 players. The central and far-from-the-net zone was mainly reached by the serve for all classes. Errors shots were similar in all classes, whilst winning shots were more frequent in C1. The current notational analysis provided a meaningful performance modelling of indicators for coaches and athletes that can be used to design training programs for each class.

Fresh-Cut Eruca Sativa Treated with Plasma Activated Water (PAW): Evaluation of Antioxidant Capacity, Polyphenolic Profile and Redox Status in Caco2 Cells.

Plasma Activated Water (PAW) has recently emerged as a promising non-chemical and non-thermal technology for the microbial decontamination of food. However, its use as a replacement for conventional disinfection solutions needs further investigation, as the impact of reactive species generated by PAW on nutritional food quality, toxicology, and safety is still unclear. The purpose of this study is to investigate how treatment with PAW affects the health-promoting properties of fresh-cut rocket salad (Eruca sativa). Therefore, the polyphenolic profile and antioxidant activity were evaluated by a combination of UHPLC-MS/MS and in vitro assays. Moreover, the effects of polyphenolic extracts on cell viability and oxidative status in Caco2 cells were assessed. PAW caused a slight reduction in the radical scavenging activity of the amphiphilic fraction over time but produced a positive effect on the total phenolic content, of about 70% in PAW-20, and an increase in the relative percentage (about 44-50%) of glucosinolate. Interestingly, the PAW polyphenol extract did not cause any cytotoxic effect and caused a lower imbalance in the redox status compared to an untreated sample. The obtained results support the use of PAW technology for fresh-cut vegetables to preserve their nutritional properties.

Antioxidant and pro-oxidant phytochemicals in ultrasound and microwave assisted extracts from hop cones: a statistical modelling approach.

The present study investigated the relationships between different green extracts from hop cones (HGEs) and their cytoprotective/cytotoxic effects on human cultured colonocytes, using a multivariate statistical approach. HGEs were obtained by ultrasound (US) and microwave (MW) assisted extraction, using food grade solvents (ethanol and ethanol : water = 50 : 50 mixture). Their chemical fingerprinting showed the presence of 21 bioactive compounds belonging to the classes of polyphenols, prenylcalcones and floroacylglucinols, which were more abundant in MW ethanolic extracts. All the extracts, except for the US hydroalcoholic one, exerted a cytotoxic effect in a dose-dependent manner. HGEs did not alter the cellular redox status at low doses, while at the highest concentrations considered they displayed a pro-oxidant or antioxidant activity. Chemometric analysis revealed the compounds most correlated with cellular toxicity and/or ROS production and that the differences observed in Caco2 cells could be adequately explained by 2D statistical models including inhibitor-promoting agent pairs.

Extracellular clusterin limits the uptake of α-synuclein fibrils by murine and human astrocytes.

The progressive neuropathological damage seen in Parkinson's disease (PD) is thought to be related to the spreading of aggregated forms of α-synuclein. Clearance of extracellular α-synuclein released by degenerating neurons may be therefore a key mechanism to control the concentration of α-synuclein in the extracellular space. Several molecular chaperones control misfolded protein accumulation in the extracellular compartment. Among these, clusterin, a glycoprotein associated with Alzheimer's disease, binds α-synuclein aggregated species and is present in Lewy bodies, intraneuronal aggregates mainly composed by fibrillary α-synuclein. In this study, using murine primary astrocytes with clusterin genetic deletion, human-induced pluripotent stem cell (iPSC)-derived astrocytes with clusterin silencing and two animal models relevant for PD we explore how clusterin affects the clearance of α-synuclein aggregates by astrocytes. Our findings showed that astrocytes take up α-synuclein preformed fibrils (pffs) through dynamin-dependent endocytosis and that clusterin levels are modulated in the culture media of cells upon α-synuclein pffs exposure. Specifically, we found that clusterin interacts with α-synuclein pffs in the extracellular compartment and the clusterin/α-synuclein complex can be internalized by astrocytes. Mechanistically, using clusterin knock-out primary astrocytes and clusterin knock-down hiPSC-derived astrocytes we observed that clusterin limits the uptake of α-synuclein pffs by cells. Interestingly, we detected increased levels of clusterin in the adeno-associated virus- and the α-synuclein pffs- injected mouse model, suggesting a crucial role of this chaperone in the pathogenesis of PD. Overall, our observations indicate that clusterin can limit the uptake of extracellular α-synuclein aggregates by astrocytes and, hence, contribute to the spreading of Parkinson pathology.

The Down-Regulation of Clusterin Expression Enhances the αSynuclein Aggregation Process.

Parkinson's Disease (PD) is a progressive neurodegenerative disease characterized by the presence of proteinaceous aggregates of αSynuclein (αSyn) in the dopaminergic neurons. Chaperones are key components of the proteostasis network that are able to counteract αSyn's aggregation, as well as its toxic effects. Clusterin (CLU), a molecular chaperone, was consistently found to interfere with Aß aggregation in Alzheimer's Disease (AD). However, its role in PD pathogenesis has yet to be extensively investigated. In this study, we assessed the involvement of CLU in the αSyn aggregation process by using SH-SY5Y cells stably overexpressing αSyn (SH-Syn). First, we showed that αSyn overexpression caused a strong increase in CLU expression without affecting levels of Hsp27, Hsp70, and Hsp90, which are the chaperones widely recognized to counteract αSyn burden. Then, we demonstrated that αSyn aggregation, induced by proteasome inhibition, determines a strong increase of CLU in insoluble aggregates. Remarkably, we revealed that CLU down-regulation results in an increase of αSyn aggregates in SH-Syn without significantly affecting cell viability and the Unfolded Protein Response (UPR). Furthermore, we demonstrated the direct molecular interaction between CLU and αSyn via a co-immunoprecipitation (co-IP) assay. All together, these findings provide incontrovertible evidence that CLU is an important player in the response orchestrated by the cell to cope with αSyn burden.

Clusterin Silencing in Prostate Cancer Induces Matrix Metalloproteinases by an NF-κB-Dependent Mechanism.

Clusterin (CLU) is a stress-activated glycoprotein, whose expression is altered both in inflammation and cancer. Previously, we showed that abrogation of CLU expression in cancer-prone mice (TRAMP) results in the enhancement of tumor spreading and homing, concomitant with an enhanced expression of NF-κB. In the present paper, we carried out an extensive experimental work by utilizing microarray gene expression data, as well as in vitro and in vivo models of prostate cancer (PCa). Our results demonstrated that (i) CLU expression is significantly downregulated in human PCa and inversely correlates with the expression of p65 in metastases; (ii) CLU overexpression in PCa cells reduces the Ser536 phosphorylation of p65, inhibits NF-κB nuclear translocation, and reduces the transcription of matrix metalloproteinase-9 and metalloproteinase-2 (MMP-9 and MMP-2). Conversely, CLU silencing promotes NF-κB activation and transcriptional upregulation of MMP-9; and (iii) expression and activity of MMP-2 and MMP-9 are increased in CLU-/- mice (CLUKO) and in TRAMP/CLUKO mice in comparison to their relative Clu+/+ littermates. Taken together, our data support the hypothesis that CLU downregulation, an early and relevant event in PCa onset, may inhibit NF-κB activation and limit the execution of a transcriptional program that favor the disease progression towards a metastatic stage.

Groin pain in athletes and non-interventional rehabilitative treatment: a systematic review.

INTRODUCTION: Groin pain is a common complaint both in high-performance and recreational athletes. Diagnosis is based on the patient's history and physical examination. Imaging assessments are often considered to exclude other pathologies. To date there is no strong evidence to support conservative or surgical treatment options. The purpose of this study is to shed light on the more effective non-interventional rehabilitative treatments for the management of groin pain in athletes and, if possible, provide guidelines useful for clinical practice. EVIDENCE ACQUISITION: The following electronic databases were searched: PubMed, Physiotherapy Evidence Database (PEDro), Scopus, Web of Science, Google and Google Scholar. Databases were investigated from January 1997 until March 2017. EVIDENCE SYNTHESIS: The results reported in the randomized clinical trial studies highlight that active treatment is better than passive treatment to improve clinical signs of groin pain. Comparing the active strategy with multi-modal treatment the latter allows a faster return to sport activity. Although the evidence remains poor, all the included literature highlights that an integrated strategy which combines active and passive treatment, the assessment of perceived pain, a return to running program and specific-sport exercises is an effective strategy for management of groin pain in athletes. CONCLUSIONS: Although we shed some light on common key aspects able to improve the typical signs of groin pain, on the basis of available data we were unable to provide practice guidelines. Further studies are necessary to set the best treatment algorithm for the management of groin pain in athletes.

Neuromuscular recovery in ACL reconstruction with Bone-Tendon-Patellar-Bone and Semitendinosus-Gracilis autograft.

OBJECTIVE: Several different types of grafts have been used in ACL rupture. The purpose of the study was to compare the recovery of lower limbs muscle strength and proprioception in athletes, who underwent ACL reconstruction with Bone-Patellar-Tendon-Bone (BPTB) versus semitendinosus and gracilis (HS) autografts. METHODS: We enrolled 30 male amateur athletes. Each patient was evaluated by isokinetic test, triaxial accelerometer test and balance test with stabilometric platform. Isokinetic test evaluated quadriceps and hamstrings Peak Torque. Accelerometer test evaluated squat jump test (SJT) and stiffness test (ST). The recording on the balance platform was performed with open and closed eyes and evaluated medio-lateral and anterior-posterior pathways. RESULTS: 30 patients were selected (15 in group BPTB and 15 in group HS). In SJT we noticed a statistically significant difference in height of jump in the involved side in favour of Group BPTB (p=0.037) and not significant difference in the other parameters. In the ST, we did not observe significant statistical differences in the parameters of the test. The stabilometric platform data and isokinetic peak torque parameters did not show a significant difference. DISCUSSION: Little high quality researches are available to help determine when patients can safely return to full activity and sport. Included evaluation criteria were a combination of factors regarding knee motion, muscles strength and neuromuscular function. CONCLUSION: In our study, despite a not full recovery of explosive strength in HS group , the balance and the other parameters after one year are comparable between the two graft. In our findings there isn't clinical difference between the two grafts. We suggest that the evaluation of explosive strength and proprioception are the priority parameters in neuromuscular recovery after ACL reconstruction.

Plantar fasciitis in athletes: diagnostic and treatment strategies. A systematic review.

BACKGROUND: Plantar fasciitis (PF) is reported in different sports mainly in running and soccer athletes. Purpose of this study is to conduct a systematic review of published literature concerning the diagnosis and treatment of PF in both recreational and élite athletes. The review was conducted and reported in accordance with the PRISMA statement. METHODS: The following electronic databases were searched: PubMed, Cochrane Library and Scopus. As far as PF diagnosis, we investigated the electronic databases from January 2006 to June 2016, whereas in considering treatments all data in literature were investigated. RESULTS: For both diagnosis and treatment, 17 studies matched inclusion criteria. The results have highlighted that the most frequently used diagnostic techniques were Ultrasonography and Magnetic Resonance Imaging. Conventional, complementary, and alternative treatment approaches were assessed. CONCLUSIONS: In reviewing literature, we were unable to find any specific diagnostic algorithm for PF in athletes, due to the fact that no different diagnostic strategies were used for athletes and non-athletes. As for treatment, a few literature data are available and it makes difficult to suggest practice guidelines. Specific studies are necessary to define the best treatment algorithm for both recreational and élite athletes. LEVEL OF EVIDENCE: Ib.

Green Tea Catechins for Prostate Cancer Prevention: Present Achievements and Future Challenges.

Green tea catechins (GTCs) are a family of chemically related compounds usually classified as antioxidant molecules. Epidemiological evidences, supported by interventional studies, highlighted a more than promising role for GTCs in human prostate cancer (PCa) chemoprevention. In the last decades, many efforts have been made to gain new insights into the mechanism of action of GTCs. Now it is clear that GTCs' anticancer action can no longer be simplistically limited to their direct antioxidant/pro-oxidant properties. Recent contributions to the advancement of knowledge in this field have shown that GTCs specifically interact with cellular targets, including cell surface receptors, lipid rafts, and endoplasmic reticulum, modulate gene expression through direct effect on transcription factors or indirect epigenetic mechanisms, and interfere with intracellular proteostasis at various levels. Many of the effects observed in vitro are dose and cell context dependent and take place at concentrations that cannot be achieved in vivo. Poor intestinal absorption together with an extensive systemic and enteric metabolism influence GTCs' bioavailability through still poorly understood mechanisms. Recent efforts to develop delivery systems that increase GTCs' overall bioavailability, by means of biopolymeric nanoparticles, represent the main way to translate preclinical results in a real clinical scenario for PCa chemoprevention.

Systematic review on strength training in Parkinson's disease: an unsolved question.

The purpose of this study was to investigate the effectiveness of strength training, performed against a different resistance from body weight, in improving motor and nonmotor symptoms in patients with Parkinson's disease (PD). The following electronic databases were searched: PubMed, Physiotherapy Evidence Database, Cochrane Central Register of Controlled Trials, Scopus, and Web of Science. The review was conducted and reported in accordance with the PRISMA statement. Thirteen high-quality randomized controlled trials were included. Strength training performed against external resistance is well tolerated and appears to be a suitable physical activity to improve both physical parameters and quality of life parameters of PD subjects. However, although the study intervention included strength training, only a few selected studies assessed the improvement of muscle strength. Despite the encouraging results, it is difficult to establish a correlation between strength training and the improvements made. Our review highlights the lack of common intent in terms of study design and the presence of different primary and secondary outcomes. Accordingly, further studies are needed to support the beneficial effects of different types of strength training in PD subjects and to underline the superiority of strength training in PD patients with respect to other training.

The Structure and Function of a Microbial Allantoin Racemase Reveal the Origin and Conservation of a Catalytic Mechanism.

The S enantiomer of allantoin is an intermediate of purine degradation in several organisms and the final product of uricolysis in nonhominoid mammals. Bioinformatics indicated that proteins of the Asp/Glu racemase superfamily could be responsible for the allantoin racemase (AllR) activity originally described in Pseudomonas species. In these proteins, a cysteine of the catalytic dyad is substituted with glycine, yet the recombinant enzyme displayed racemization activity with a similar efficiency (kcat/KM ≈ 5 × 104 M-1 s-1) for the R and S enantiomers of allantoin. The protein crystal structure identified a glutamate residue located three residues downstream (E78) that can functionally replace the missing cysteine; the catalytic role of E78 was confirmed by site-directed mutagenesis. Allantoin can undergo racemization through formation of a bicyclic intermediate (faster) or proton exchange at the chiral center (slower). By monitoring the two alternative mechanisms by 13C and 1H nuclear magnetic resonance, we found that the velocity of the faster reaction is unaffected by the enzyme, whereas the velocity of the slower reaction is increased by 7 orders of magnitude. Protein phylogenies trace the origin of the racemization mechanism in enzymes acting on glutamate, a substrate for which proton exchange is the only viable reaction mechanism. This mechanism was inherited by allantoin racemase through divergent evolution and conserved in spite of the substitution of catalytic residues.

Effect of Cold Plasma Treatment on the Functional Properties of Fresh-Cut Apples.

Atmospheric double-barrier discharge (DBD) plasma technology is a promising tool in the food industry as an alternative to traditional food preservation methods. However, the effect of the reactive species generated during the treatment on the content of bioactive compounds in food is still little studied, and there are no data concerning potential deleterious effects of DBD-treated foods on human cells. Some functional properties of DBD-treated minimally processed Pink Lady apples were evaluated in comparison with untreated samples through different in vitro and ex vivo tests. Plasma treatment caused only a slight reduction of antioxidant content and antioxidant capacity (up to 10%), mainly limited to the amphiphilic fraction. Noteworthy, treated apple polyphenol extracts did not reduce cell viability and did not suppress the beneficial physiological cell response to oxidative stress in terms of reactive oxygen species production and phase II enzyme activation in human cultured colonocytes.

Long term effect of selective muscle strengthening in athletes with patellofemoral pain syndrome.

BACKGROUND AND AIM OF THE WORK: The purpose of the study was to examine the long term effects of a selective muscle strengthening program in reducing pain and improving knee function and strength in athletes with Patellofemoral Pain Syndrome. METHODS: A total of one hundred and thirty four athletes were enrolled in the study. All patients were evaluated with Isokinetic Test, Cincinnati Knee Rating System and Visual Analogue Scale. The selective muscle strengthening consisted of 8 weeks of exercises performed 3 times in the first 4 weeks and twice in the last 4 weeks. The muscle strengthening program was performed between 30-90° of knee flexion. During the first 4-weeks treatment we used closed kinetic chain exercises with 3 sets of 8 repetitions at 80% of maximum load. In the last 4-weeks we added open kinetic chain exercises at 70% of maximum load with 3 sets and 10 repetitions to improve the resistance. RESULTS: Analyzing data at the beginning and at the end of the treatment for Isokinetic test, Cincinnati and Visual Analogue Scale we observed a significant scores improvement. At 1 year follow-up the clinical improvements were maintained and everyone followed the recommended program because did not perform the maintenance program. At 2 years follow-up no athletes presented relapses; only four patients were excluded from program. CONCLUSIONS: We believe that our program of selective muscle strengthening should resolve pain and improve knee function and strength as results in obtained scores and could be critical to avoid painful relapses.

EGCG antagonizes Bortezomib cytotoxicity in prostate cancer cells by an autophagic mechanism.

The proteasome inhibitors Bortezomib (BZM) and MG132 trigger cancer cell death via induction of endoplasmic reticulum (ER) stress and unfolded protein response. Epigallocatechin gallate (EGCG), the most bioactive green tea polyphenol, is known to display strong anticancer properties as it inhibits proteasome activity and induces ER stress. We investigated whether combined delivery of a proteasome inhibitor with EGCG enhances prostate cancer cell death through increased induction of ER stress. Paradoxically, EGCG antagonized BZM cytotoxicity even when used at low concentrations. Conversely, the MG132 dose-response curve was unaffected by co-administration of EGCG. Moreover, apoptosis, proteasome inhibition and ER stress were inhibited in PC3 cells simultaneously treated with BZM and EGCG but not with a combination of MG132 and EGCG; EGCG enhanced autophagy induction in BZM-treated cells only. Autophagy inhibition restored cytotoxicity concomitantly with CHOP and p-eIF2α up-regulation in cells treated with BZM and EGCG. Overall, these findings demonstrate that EGCG antagonizes BZM toxicity by exacerbating the activation of autophagy, which in turn mitigates ER stress and reduces CHOP up-regulation, finally protecting PC3 cells from cell death.

Distinct promoters, subjected to epigenetic regulation, drive the expression of two clusterin mRNAs in prostate cancer cells.

The human clusterin (CLU) gene codes for several mRNAs characterized by different sequences at their 5' end. We investigated the expression of two CLU mRNAs, called CLU 1 and CLU 2, in immortalized (PNT1a) and tumorigenic (PC3 and DU145) prostate epithelial cells, as well as in normal fetal fibroblasts (WI38) following the administration of the epigenetic drugs 5-aza-2'-deoxycytidine (AZDC) and trichostatin A (TSA) given either as single or combined treatment (AZDC-TSA). Our experimental evidences show that: a) CLU 1 is the most abundant transcript variant. b) CLU 2 is expressed at a low level in normal fibroblasts and virtually absent in prostate cancer cells. c) CLU 1, and to a greater extent CLU 2 expression, increased by AZDC-TSA treatment in prostate cancer cells. d) Both CLU 1 and CLU 2 encode for secreted CLU. e) P2, a novel promoter that overlaps the CLU 2 Transcription Start Site (TSS), drives CLU 2 expression. f) A CpG island, methylated in prostate cancer cells and not in normal fibroblasts, is responsible for long-term heritable regulation of CLU 1 expression. g) ChIP assay of histone tail modifications at CLU promoters (P1 and P2) shows that treatment of prostate cancer cells with AZDC-TSA causes enrichment of Histone3(Lys9)acetylated (H3K9ac) and reduction of Histone3(Lys27)trimethylated (H3K27me3), inducing active transcription of both CLU variants. In conclusion, we show for the first time that the expression of CLU 2 mRNA is driven by a novel promoter, P2, whose activity responds to epigenetic drugs treatment through changes in histone modifications.

Polyphenon E(R), a standardized green tea extract, induces endoplasmic reticulum stress, leading to death of immortalized PNT1a cells by anoikis and tumorigenic PC3 by necroptosis.

Increasing doses of Polyphenon E®, a standardized green tea extract, were given to PNT1a and PC3 prostate epithelial cells mimicking initial and advanced stages of prostate cancer (PCa), respectively. Cell death occurred in both cell lines, with PNT1a being more sensitive [half-maximal inhibitory concentration (IC50) = 35 µg/ml] than PC3 (IC50 = 145 µg/ml) to Polyphenon E®. Cell cycle arrest occurred at G0/G1 checkpoint for PNT1a, and G2/M for PC3 cells. Endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) occurred in both cell lines, with each exhibiting different timing in response to Polyphenon E®. Autophagy was transiently activated in PNT1a cells within 12 h after treatment as a survival response to overcome ERS; then activation of caspases and cleavage of poly (ADP ribose) polymerase 1 occurred, committing cells to anoikis death. Polyphenon E® induced severe ERS in PC3 cells, causing a dramatic enlargement of the ER; persistent activation of UPR produced strong upregulation of GADD153/CHOP, a key protein of ERS-mediated cell death. Thereafter, GADD153/CHOP activated Puma, a BH3-only protein, committing cells to necroptosis, a programmed caspase-independent mechanism of cell death. Our results provide a foundation for the identification of novel targets and strategies aimed at sensitizing apoptosis-resistant cells to alternative death pathways.

Isoform identification, recombinant production and characterization of the allergen lipid transfer protein 1 from pear (Pyr c 3).

Non-specific lipid transfer proteins belonging to LTP1 family represent the most important allergens for non pollen-related allergies to Rosaceae fruits in the Mediterranean area. Peach LTP1 (Pru p 3) is a major allergen and is considered the prototypic allergenic LTP. On the contrary, pear allergy without pollinosis seems to be under-reported when compared to other Rosaceae fruits suggesting that the as-yet-uncharacterized pear LTP1 (Pyr c 3) has in vivo a low allergenicity. We report here on the identification of four cDNAs encoding for LTP1 in pear fruits. The two isoforms exhibiting amino acid sequences most similar to those of peach and apple homologues were obtained as recombinant proteins. Such isoforms exhibited CD spectra and lipid binding ability typical of LTP1 family. Moreover, pear LTP1 mRNA was mainly found in the peel, as previously shown for other Rosaceae fruits. By means of IgE ELISA assays a considerable immunoreactivity of these proteins to LTP-sensitive patient sera was detected, even though allergic reactions after ingestion of pear were not reported in the clinical history of the patients. Finally, the abundance of LTP1 in protein extracts from pear peel, in which LTP1 from Rosaceae fruits is mainly confined, was estimated to be much lower as compared to peach peel. Our data suggest that the two isoforms of pear LTP1 characterized in this study possess biochemical features and IgE-binding ability similar to allergenic LTPs. Their low concentrations in pear might be the cause of the low frequency of LTP-mediated pear allergy.

Probing the evolution of hydroxyisourate hydrolase into transthyretin through active-site redesign.

5-Hydroxyisourate hydrolase (HIUase) and transthyretin (TTR) are closely related phylogenetically and structurally, while performing quite different functions. The former catalyzes the hydrolysis of 5-hydroxyisourate within the urate degradation pathway, and the latter is a carrier protein involved in the extracellular transport of thyroid hormones and in the cotransport of retinol. The evolution of HIUase into TTR represents a remarkable example of adaptation of a new function by active-site modification of an enzyme. On the basis of phylogenetic reconstructions and structural comparison of HIUase and TTR, two mutations (Y116T and I16A) were likely to be crucial events in order to induce, after a gene duplication event, the conversion of the enzyme into a binding protein. By rational reshaping of the active sites of HIUase and functional analyses of its mutant forms, we have provided insights into how its neofunctionalization could be achieved. We show here that the two mutations at the active sites of HIUase open up the two ends of the channel that transverses the entire tetrameric protein, generating two cavities accessible to the thyroxine molecule and abrogating, at the same time, the enzymatic activity. Our data indicate that a small number of critical mutations affecting the active site of an enzyme may be sufficient to generate a drastically different function, while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins.