The Mechanism of Action of L-Tyrosine Derivatives against Chikungunya Virus Infection In Vitro Depends on Structural Changes.

Although the disease caused by chikungunya virus (CHIKV) is of great interest to public health organizations around the world, there are still no authorized antivirals for its treatment. Previously, dihalogenated anti-CHIKV compounds derived from L-tyrosine (dH-Y) were identified as being effective against in vitro infection by this virus, so the objective of this study was to determine the mechanisms of its antiviral action. Six dH-Y compounds (C1 to C6) dihalogenated with bromine or chlorine and modified in their amino groups were evaluated by different in vitro antiviral strategies and in silico tools. When the cells were exposed before infection, all compounds decreased the expression of viral proteins; only C4, C5 and C6 inhibited the genome; and C1, C2 and C3 inhibited infectious viral particles (IVPs). Furthermore, C1 and C3 reduce adhesion, while C2 and C3 reduce internalization, which could be related to the in silico interaction with the fusion peptide of the E1 viral protein. Only C3, C4, C5 and C6 inhibited IVPs when the cells were exposed after infection, and their effect occurred in late stages after viral translation and replication, such as assembly, and not during budding. In summary, the structural changes of these compounds determine their mechanism of action. Additionally, C3 was the only compound that inhibited CHIKV infection at different stages of the replicative cycle, making it a compound of interest for conversion as a potential drug.

Photo-tuneable protein nitration by sensitiser tris(bipyridine)-Ruthenium(II) chloride complex.

Post-translational modifications (PTMs) of proteins are a diverse source of variability that impacts on their functions, localisation, regulation, and lifetime. However, one of the main pitfalls in their study is that they appear in rather low frequencies and/or are only transiently observed. To overcome this issue and ease the study in vitro of stress-related protein PTMs, several methods have been proposed to model stress conditions and chemically introduce them. These techniques employ the combination of peroxides with transition metal ions or haem-containing proteins, as well as other possibilities such as peroxy radicals or UV radiation. However, their control, reproducibility and undesired secondary reactions that reduce the process yield are often a matter of concern. Here we introduce a photo-tuneable method that selectively targets nitration of aromatic residues. We initially present the adaptation of an oxidation method based on the photosensitiser tris(2,2'-bipyridine)-Ruthenium(II) chloride complex and ammonium persulfate, in which we employ an alternative radical neutralisation/trapping pathway that uses nitrite ions for the nitration of free l-Tyrosine and L-Tryptophan amino acids. After analysing the effect of several factors, we report the application of the photo-tuneable protein nitration (PTPN) method to four different model proteins in which we evaluate the nitration and oxidation of residues in each case. A mass spectrometry label-free quantitation of Tyr and Trp nitration is also described in order to compare the degree of modification and the accessibility of these residues. The method described could be employed to scale up the production of proteins with a selected range of oxidative PTMs for their characterisation, the assessment of their pathophysiological roles, and the development of detection and quantification methods to validate these PTMs as novel biomarkers associated with oxidative stress-related pathologies, such as in cardiovascular or neurodegenerative diseases.

Photosensitized Dimerization of Tyrosine: The Oxygen Paradox.

In electron-transfer initiated photosensitization processes, molecular oxygen (O2 ) is not involved in the first bimolecular event, but almost always participates in subsequent steps giving rise to oxygenated products. An exception to this general behavior is the photosensitized dimerization of tyrosine (Tyr), where O2 does not participate as a reactant in any step of the pathway yielding Tyr dimers (Tyr2 ). In the pterin (Ptr) photosensitized oxidation of Tyr, O2 does not directly participate in the formation of Tyr2 and quenches the triplet excited state of Ptr, the reactive species that initiates the process. However, O2 is necessary for the dimerization, phenomenon that we have named as the oxygen paradox. Here, we review the literature on the photosensitized formation of Tyr2 and present results of steady-state and time resolved experiments, in search of a mechanistic model to explain the contradictory role of O2 in this photochemical reaction system.

A novel violet fluorescent protein contains a unique oxidized tyrosine as the simplest chromophore ever reported in fluorescent proteins.

We describe an engineered violet fluorescent protein from the lancelet Branchiostoma floridae (bfVFP). This is the first example of a GFP-like fluorescent protein with a stable fluorescent chromophore lacking an imidazolinone ring; instead, it consists of oxidized tyrosine 68 flanked by glycine 67 and alanine 69. bfVFP contains the simplest chromophore reported in fluorescent proteins and was generated from the yellow protein lanFP10A2 by two synergetic mutations, S148H and C166I. The chromophore structure was confirmed crystallographically and by high-resolution mass spectrometry. The photophysical characteristics of bfVFP (323/430 nm, quantum yield 0.33, and Ec 14,300 M-1  cm-1 ) make it potentially useful for multicolor experiments to expand the excitation range of available FP biomarkers and Förster resonance energy transfer with blue and cyan fluorescent protein acceptors.

Exploration of the Activation Mechanism of the Epigenetic Regulator MLL3: A QM/MM Study.

The mixed lineage leukemia 3 or MLL3 is the enzyme in charge of the writing of an epigenetic mark through the methylation of lysine 4 from the N-terminal domain of histone 3 and its deregulation has been related to several cancer lines. An interesting feature of this enzyme comes from its regulation mechanism, which involves its binding to an activating dimer before it can be catalytically functional. Once the trimer is formed, the reaction mechanism proceeds through the deprotonation of the lysine followed by the methyl-transfer reaction. Here we present a detailed exploration of the activation mechanism through a QM/MM approach focusing on both steps of the reaction, aiming to provide new insights into the deprotonation process and the role of the catalytic machinery in the methyl-transfer reaction. Our finding suggests that the source of the activation mechanism comes from conformational restriction mediated by the formation of a network of salt-bridges between MLL3 and one of the activating subunits, which restricts and stabilizes the positioning of several residues relevant for the catalysis. New insights into the deprotonation mechanism of lysine are provided, identifying a valine residue as crucial in the positioning of the water molecule in charge of the process. Finally, a tyrosine residue was found to assist the methyl transfer from SAM to the target lysine.

Structural features of the plant N-recognin ClpS1 and sequence determinants in its targets that govern substrate selection.

In the N-degron pathway of protein degradation of Escherichia coli, the N-recognin ClpS identifies substrates bearing N-terminal phenylalanine, tyrosine, tryptophan, or leucine and delivers them to the caseinolytic protease (Clp). Chloroplasts contain the Clp system, but whether chloroplastic ClpS1 adheres to the same constraints is unknown. Moreover, the structural underpinnings of substrate recognition are not completely defined. We show that ClpS1 recognizes canonical residues of the E. coli N-degron pathway. The residue in second position influences recognition (especially in N-terminal ends starting with leucine). N-terminal acetylation abrogates recognition. ClpF, a ClpS1-interacting partner, does not alter its specificity. Substrate binding provokes local remodeling of residues in the substrate-binding cavity of ClpS1. Our work strongly supports the existence of a chloroplastic N-degron pathway.

Intracellular Progesterone Receptor and cSrc Protein Working Together to Regulate the Activity of Proteins Involved in Migration and Invasion of Human Glioblastoma Cells.

Glioblastomas are the most common and aggressive primary brain tumors in adults, and patients with glioblastoma have a median survival of 15 months. Some alternative therapies, such as Src family kinase inhibitors, have failed presumably because other signaling pathways compensate for their effects. In the last ten years, it has been proven that sex hormones such as progesterone (P4) can induce growth, migration, and invasion of glioblastoma cells through its intracellular progesterone receptor (PR), which is mostly known for its role as a transcription factor, but it can also induce non-genomic actions. These non-classic actions are, in part, a consequence of its interaction with cSrc, which plays a significant role in the progression of glioblastomas. We studied the relation between PR and cSrc, and its effects in human glioblastoma cells. Our results showed that P4 and R5020 (specific PR agonist) activated cSrc protein since both progestins increased the p-cSrc (Y416)/cSrc ratio in U251 and U87 human glioblastoma derived cell lines. When siRNA against the PR gene was used, the activation of cSrc by P4 was abolished. The co-immunoprecipitation assay showed that cSrc and PR interact in U251 cells. P4 treatment also promoted the increase in the p-Fak (Y397) (Y576/577)/Fak and the decrease in p-Paxillin (Y118)/Paxillin ratio, which are significant components of the focal adhesion complex and essential for migration and invasion processes. A siRNA against cSrc gene blocked the increase in the p-Fak (Y576/Y577)/Fak ratio and the migration induced by P4, but not the decrease in p-Paxillin (Y118)/Paxillin ratio. We analyzed the potential role of cSrc over PR phosphorylation in three databases, and one putative tyrosine residue in the amino acid 87 of PR was found. Our results showed that P4 induces the activation of cSrc protein through its PR. The latter and cSrc could interact in a bidirectional mode for regulating the activity of proteins involved in migration and invasion of glioblastomas.

Nickel(II) complexes based on L-amino-acid-derived ligands: synthesis, characterization and study of the role of the supramolecular structure in carbon dioxide capture.

The formation of the symmetrical µ3-carbonate-bridged self-assembled trinuclear NiII complex Na2{[Ni(LO)2(H2O)]3(µ3-CO3)} (LO is the carboxylate anion of a L-tyrosine derivative), involves atmospheric CO2 uptake. The asymmetric unit of the complex comprises an octahedral coordination for the NiII with two L-tyrosine-based ligands, a water molecule and one O atom of the carbonate bridge. The Ni3-µ3-CO3 core in this compound is the first reported of this kind according to the Cambridge Structural Database (CSD). The supramolecular structure is mainly sustained by hydrogen bonds developed by the phenolic functionality of the L-tyrosine moiety of one ligand and the carboxylate group of a neighbouring ligand. The crystal packing is then characterized by three interpenetrated supramolecular helices associated with a diastereoisomer of the type R-supP, which is essential for the assembly process. Magnetic susceptibility and magnetization data support weak ferromagnetic exchange interactions within the novel Ni3-µ3-CO3 core. The NiII complex obtained under the same synthetic conditions but using the analogous ligand derived from the amino acid L-phenylalanine instead of L-tyrosine gives rise to to a mononuclear octahedral system. The results obtained for the different complexes demonstrate the role of the supramolecular structure regarding the CO2 uptake property for these NiII-amino-acid-based systems.

Tyr-nitration in maize CDKA;1 results in lower affinity for ATP binding.

Cyclin-dependent kinase A (CDKA) is a key component for cell cycle progression. The catalytic kinase activity depends on the protein's ability to form an active complex with cyclins and on phosphoregulatory mechanisms. Cell cycle arrest and plant growth impairment under abiotic stress have been linked to different molecular processes triggered by increased levels of reactive oxygen and nitrogen species (ROS and RNS). Among these, posttranslational modifications (PTMs) of key proteins such as CDKA;1 may be of significance. Herein, isolated maize embryo axes were subjected to sodium nitroprusside (SNP) as an inductor of nitrosative conditions to evaluate if CDKA;1 protein was a target for RNS. A high degree of protein nitration was detected; this included the specific Tyr-nitration of CDKA;1. Tyr15 and Tyr19, located at the ATP-binding site, were the selective targets for nitration according to both in silico analysis using the predictive software GPS-YNO2, and in vitro mass spectrometry studies of recombinant nitrated ZmCDKA;1. Spectrofluorometric measurements demonstrated a reduction of ZmCDKA;1-NO2 affinity for ATP. From these results, we conclude that Tyr nitration in CDKA;1 could act as an active modulator of cell cycle progression during redox stress.

Differential Proliferation Effect of the Newly Synthesized Valine, Tyrosine and Tryptophan-Naphthoquinones in Immortal and Tumorigenic Cervical Cell Lines.

We previously showed that microwave assisted synthesis is the best method for the synthesis of naphthoquinone amino acid and chloride-naphthoquinone amino acid derivatives by a complete evaluation of reaction conditions such as stoichiometry, bases, and pH influence. Following the same strategy, we synthesized chloride and non-chloride tyrosine, valine, and tryptophan-naphthoquinones achieving 85-95%, 80-92%, and 91-95% yields, respectively. The cyclic voltammetry profiles showed that both series of naphthoquinone amino acid derivatives mainly display one redox reaction process. Overall, chloride naphthoquinone amino acid derivatives exhibited redox potential values (E1/2) more positive than non-chloride compounds. The six newly synthesized compounds were tested in HPV positive and negative as well as in immortal and tumorigenic cell lines to observe the effects in different cellular context simulating precancerous and cancerous status. A dose-response was achieved to determine the IC50 of six newly synthesized compounds in SiHa (Tumorigenic and HPV16 positive), CaLo (Tumorigenic and HPV18 positive), C33-A (Tumorigenic and HPV negative) and HaCaT (Keratinocytes immortal HPV negative) cell lines. Non-chloride tryptophan-naphthoquinone (3c) and chloride tyrosine-naphthoquine (4a) effects were more potent in tumorigenic SiHa, CaLo, and C33-A cells with respect to non-tumorigenic HaCaT cells. Interestingly, there seems to be a differential effect in non-chloride and chloride naphthoquinone amino acid derivatives in tumorigenic versus non tumorigenic cells. Considering all naphthoquinone amino acid derivatives that our group synthesized, it seems that hydrophobic and aromatic amino acids have the greatest effect on cell proliferation inhibition. These results show promising compounds for cervical cancer treatment.

3-Nitrotyrosine and related derivatives in proteins: precursors, radical intermediates and impact in function.

Oxidative post-translational modification of proteins by molecular oxygen (O2)- and nitric oxide (•NO)-derived reactive species is a usual process that occurs in mammalian tissues under both physiological and pathological conditions and can exert either regulatory or cytotoxic effects. Although the side chain of several amino acids is prone to experience oxidative modifications, tyrosine residues are one of the preferred targets of one-electron oxidants, given the ability of their phenolic side chain to undergo reversible one-electron oxidation to the relatively stable tyrosyl radical. Naturally occurring as reversible catalytic intermediates at the active site of a variety of enzymes, tyrosyl radicals can also lead to the formation of several stable oxidative products through radical-radical reactions, as is the case of 3-nitrotyrosine (NO2Tyr). The formation of NO2Tyr mainly occurs through the fast reaction between the tyrosyl radical and nitrogen dioxide (•NO2). One of the key endogenous nitrating agents is peroxynitrite (ONOO-), the product of the reaction of superoxide radical (O2•-) with •NO, but ONOO--independent mechanisms of nitration have been also disclosed. This chemical modification notably affects the physicochemical properties of tyrosine residues and because of this, it can have a remarkable impact on protein structure and function, both in vitro and in vivo. Although low amounts of NO2Tyr are detected under basal conditions, significantly increased levels are found at pathological states related with an overproduction of reactive species, such as cardiovascular and neurodegenerative diseases, inflammation and aging. While NO2Tyr is a well-established stable oxidative stress biomarker and a good predictor of disease progression, its role as a pathogenic mediator has been laboriously defined for just a small number of nitrated proteins and awaits further studies.

Extracellular vesicles from oviductal isthmus and ampulla stimulate the induced acrosome reaction and signaling events associated with capacitation in bovine spermatozoa.

Cells can communicate with other neighboring or distant cells through the secretion of extracellular vesicles (EV), composed of a lipid bilayer and bearing surface molecules that allow them to recognize target cells. In this way, EV induce signaling via different mechanisms, modulating the physiological state of the recipient cell. EV have been identified in both male and female reproductive fluids, however, the possible role of EV isolated from female reproductive fluids has become an emerging field only recently. It is known that ejaculated mammalian spermatozoa need to undergo physiological preparation in the female reproductive tract to fertilize the egg. EV secreted by different regions of the female tract constitute signals that may have a key role in regulating sperm functions. The aims of the present study were isolating EV from different regions of the bovine oviduct and analyzing their interaction and physiological effects on spermatozoa. Here, we report the characterization of bovine oviductal fluid EV from the isthmus and ampulla region and their effect on the induced acrosome reaction and signaling events associated with sperm capacitation. EV induced an increase in sperm protein tyrosine phosphorylation, while cell survival of cryopreserved bovine spermatozoa was maintained. We also show that EV uptake regulates the sperm calcium levels by inducing an immediate increase in the intracellular calcium concentration and sperm priming, after a pre-incubation period, of the progesterone-induced intracellular calcium rise. Our data contribute to understand the role of EV in the communication between the female reproductive tract and the sperm physiology, information that may be used to improve the efficiency of reproductive assisted technologies.

Polyacrylamide-alginate (PAAm-Alg) and phospho-L-tyrosine-linked PAAm-Alg monolithic cryogels: Purification of IgG from human serum.

In recent decades cryogels as monolithic materials have gained interest as stationary phase in chromatography for purification of biomolecules. In this study, polyacrylamide-alginate (PAAm-Alg) monolithic cryogels were prepared by cryo-copolymerization of acrylamide and alginate monomers and methylene-bisacrylamide as crosslinker to be used as a matrix in affinity chromatography for purification of proteins. Ortho-phospho-L-tyrosine (P-Tyr) was covalently attached onto PAAm-Alg cryogels via bisoxirane-activation (PAAm-Alg-Bix-P-Tyr) and both derivatized and non-derivatized cryogels were utilized for the purification of immunoglobulin G (IgG) from human serum. Cryogels were characterized by scanning electron microscopy, swelling tests, elemental analysis, FTIR, and flow dynamics. The effects of buffer systems, conductivity, and pH on IgG adsorption were studied. Through breakthrough curve analysis a dynamic capacity of 9.2 mg IgG/mL with an IgG purity of 94% was obtained (based on ELISA analysis of IgG and albumin) for PAAm-Alg-Bix-P-Tyr cryogel when human serum was diluted in 10 mmol/L NaP buffer at pH 6.0. The adsorption isotherm data were well described by the Langmuir model with value of maximum adsorption capacity of 36.12 ±â€¯3.63 mg of IgG/g for PAAm-Alg-Bix-P-Tyr. The PAAm-Alg-Bix-P-Tyr cryogel provides an attractive alternative for adsorption of IgG from human serum.

Binding of rose bengal to lysozyme modulates photooxidation and cross-linking reactions involving tyrosine and tryptophan.

This work examined the hypothesis that interactions of Rose Bengal (RB2-) with lysozyme (Lyso) might mediate type 1 photoreactions resulting in protein cross-linking even under conditions favoring 1O2 formation. UV-visible spectrophotometry, isothermal titration calorimetry (ITC), and docking analysis were employed to characterize RB2--Lyso interactions, while oxidation of Lyso was studied by SDS-PAGE gels, extent of amino acid consumption, and liquid chromatography (LC) with mass detection (employing tryptic peptides digested in H218O and H2O). Docking studies showed five interaction sites including the active site. Hydrophobic interactions induced a red shift of the visible spectrum of RB2- giving a Kd of 4.8 µM, while data from ITC studies, yielded a Kd of 0.68 µM as an average of the interactions with stoichiometry of 3.3 RB2- per Lyso. LC analysis showed a high consumption of readily-oxidized amino acids (His, Trp, Met and Tyr) located at different and diverse locations within the protein. This appears to reflect extensive damage on the protein probably mediated by a type 2 (1O2) mechanism. In contrast, docking and mass spectrometry analysis provided evidence for the generation of specific intra- (Tyr23-Tyr20) and inter-molecular (Tyr23-Trp62) Lyso cross-links, and Lyso dimer formation via radical-radical, type 1 mechanisms.

Photochemistry of tyrosine dimer: when an oxidative lesion of proteins is able to photoinduce further damage.

The tyrosine dimer (Tyr2), a covalent bond between two tyrosines (Tyr), is one of the most important modifications of the oxidative damage of proteins. This compound is increasingly used as a marker of aging, stress and pathogenesis. At physiological pH, Tyr2 is able to absorb radiation at wavelengths significantly present in the solar radiation and artificial sources of light. As a result, when Tyr2 is formed in vivo, a new chromophore appears in the proteins. Despite the biomedical importance of Tyr2, the information of its photochemical properties is limited due to the drawbacks of its synthesis. Therefore, in this work we demonstrate that at physiological pH, Tyr2 undergoes oxidation upon UV excitation yielding different products which conserve the dimeric structure. During its photodegradation different reactive oxygen species, like hydrogen peroxide, superoxide anion and singlet oxygen, are produced. Otherwise, we demonstrated that Tyr2 is able to sensitize the photodegradation of tyrosine. The results presented in this work confirm that Tyr2 can act as a potential photosensitizer, contributing to the harmful effects of UV-A radiation on biological systems.

Halogenated Tyrosine Derivatives from the Tropical Eastern Pacific Zoantharians Antipathozoanthus hickmani and Parazoanthus darwini.

In the search for bioactive marine natural products from zoantharians of the Tropical Eastern Pacific, four new tyrosine dipeptides, named valdiviamides A-D (1-4), were isolated from Antipathozoanthus hickmani, and two new tyramine derivatives, 5 and 6, from Parazoanthus darwini. The phenols of all six tyrosine derivatives are substituted by bromine and/or iodine atoms at the ortho positions of the hydroxyl. The planar structures of these aromatic alkaloids were elucidated from 1D and 2D NMR experiments in combination with HRESIMS data, and the absolute configurations of 1-4 were deduced from comparison between experimental and calculated electronic circular dichroism spectra. As halogenated tyrosine derivatives could represent chemotaxonomic markers of these genera, we decided to undertake the first chemical investigation of another species, Terrazoanthus cf. patagonichus. As expected, no halogenated metabolite was evidenced in the species, but we report herein the identification of two new zoanthoxanthin derivatives, named zoamides E (7) and F (8), from this species. Antimicrobial and cytotoxicity bioassays revealed that valdiviamide B (2) displayed moderate cytotoxicity against the HepG2 cell line with an IC50 value of 7.8 µM.

A computational investigation of the reactions of tyrosyl, tryptophanyl, and cysteinyl radicals with nitric oxide and molecular oxygen.

Proteins are main targets of oxidants in biological systems. This oxidation may occur in the protein backbone as well as in certain amino acid side chains, depending on the oxidant and amino acid intrinsic reactivity. Moreover, many enzymes are capable of generating stable amino acid radicals, such as tyrosyl, tryptophanyl and cysteinyl radicals. These species react very rapidly (many times as diffusion-controlled reactions) with relevant cellular open-shell species such as nitric oxide (·NO) or molecular oxygen (O2). The exception to this apparent rule is tyrosyl radical, that reacts at diffusion rates with ·NO, but shows very slow reactivity towards O2 (rate constant <103 M-1 s-1). In this work, we provide a comparative molecular-level description of the reaction mechanisms involved in the reactions of tyrosyl, tryptophanyl and cysteinyl radicals towards ·NO and O2, through quantum mechanics simulations which allow us to obtain relevant energetic and structural parameters, proposing a molecular explanation to this tyrosyl discrimination capability, namely, its marginal reactivity with O2.

Eficacia y seguridad de los suplementos nutricionales libres de tirosina y fenilalanina para el tratamiento de pacientes menores de 18 años con diagnóstico de tirosinemia tipo I / Efficacy and safety of tyrosine and phenylalanine free nutritional supplements for the treatment of patients under 18 years of age with diagnosis of tyrosinemia type I

INTRODUCCIÓN: La tirosinemia tipo 1 (HT-1) es una enfermedad genética infrecuente causada por la mutación del gen FAH. Esta mutación produce escasez de la enzima fumaril-aceto-acetato hidrolasa; responsable del último paso en la vía de degradación de la tirosina. El acúmulo de los metabolitos tóxicos de la tirosina interrumpe severamente el metabolismo intracelular del hígado y riñón; dañando estos órganos. La HT-1 se presenta en uno de cada 100.000 - 120.000 nacimientos alrededor del mundo. El tratamiento de los pacientes con HT-1 está dirigido a controlar los niveles plasmáticos de tirosina y evitar la formación de sus metabolitos tóxicos. El manejo nutricional reduce la ingesta de tirosina y fenilalanina mediante la dieta. El tratamiento farmacológico involucra el uso de nitisinona; la cual bloquea el segundo paso de la vía de degradación de la tirosina. De esta forma, se evita la formación de los metabolitos tóxicos de la tirosina; pero se incrementa el nivel plasmático de tirosina; lo cual produce, a su vez, molestias visuales debidas a la formación de cristales de tirosina. Para mantener el nivel de tirosina dentro del rango normal, la dieta debe estar reducida en tirosina y fenilalanina (precursor de la tirosina). Esto refuerza la importancia del manejo nutricional mediante la restricción de la ingesta de tirosina y fenilalanina en la dieta. Sin embargo, esta dieta podría no cubrir los demás requerimientos nutricionales, energéticos y proteicos de los pacientes con HT-1. Por lo tanto, los suplementos nutricionales serían necesarios para complementar la dieta. En EsSalud se dispone de suplementos nutricionales pediátricos. Sin embargo, estos no están libres de fenilalanina y tirosina. OBJETIVO: el presente dictamen preliminar expone la eficacia y seguridad de los suplementos nutricionales libres de fenilalanina y tirosina, junto con una dieta reducida en tirosina, comparado con una dieta reducida en tirosina para el tratamiento de los pacientes menores de 18 años, con diagnóstico de HT-1. METODOLOGÍA: Tras una búsqueda sistemática de literatura publicada hasta diciembre del 2018, no se encontró evidencia científica que evalúe el uso de suplementos nutricionales libres de fenilalanina y tirosina acompañados de una dieta reducida en tirosina, comparado con un manejo nutricional que involucre solo la dieta reducida en tirosina, en pacientes menores de 18 años con diagnóstico de HT-1. En su lugar, se hallaron tres artículos de recomendación tipo revisión que abordan el manejo nutricional de los pacientes con HT-1. Uno de ellos no menciona los suplementos libres de tirosina y fenilalanina. Los otros dos, mencionan que es necesario añadir estos suplementos a la dieta de todo paciente con HT-1. Sin embargo, esta afirmación no forma parte de las recomendaciones propiamente dichas, y no se mostraron las referencias de la evidencia utilizada para dar esta afirmación. RESULTADOS: Pese a la escasa evidencia científica disponible, la eficacia de los suplementos nutricionales libres de tirosina y fenilalanina tiene sustento biológico razonable. El requerimiento varía desde 3 g/kg/día, aproximadamente, en menores de 2 años hasta 1.5 g/Kg/día a los 10-14 años. Sin embargo, los requerimientos de tirosina y fenilalanina se alcanzan con pequeñas cantidades de proteína natural (0.5 a 1.0 g/Kg/día). Al restringir la ingesta de proteína natural (para evitar el exceso de tirosina y fenilalanina en pacientes con HT-1), se crea un déficit para cubrir el requerimiento proteico global. Si este déficit no se cubre, el paciente tendrá problemas en el crecimiento y desarrollo psicomotor. Por lo tanto, la inclusión de suplementos nutricionales libres de tirosina y fenilalanina en la dieta de los pacientes HT-1 ayuda a cubrir sus requerimientos proteicos, manteniendo las concentraciones plasmáticas de tirosina en el rango saludable. En línea con lo mencionado, el experto en pediatría de la institución, afirma que los suplementos nutricionales libres de tirosina y fenilalanina permiten el adecuado crecimiento y desarrollo neurológico mientras evita la aparición de problemas visuales. Por lo tanto, el experto en pediatría opina que estos suplementos nutricionales son un complemento terapéutico necesario para el tratamiento dietético. Esta posición coincide con la opinión de los expertos internacionales y las revisiones presentadas en el presente dictamen preliminar. CONCLUSIÓN: Con respecto a lo expuesto previamente, el equipo técnico del IETSI valoró los siguientes aspectos: i) La HT-1 es una enfermedad hereditaria, infrecuente, que afecta la calidad de vida del paciente desde edades muy tempranas y está asociada a una alta mortalidad si no es tratada adecuadamente ii) En el contexto de EsSalud existe un vacío terapéutico para la población objetivo, iii) Aunque la evidencia disponible actualmente es limitada, algunas guías mencionan el uso de suplementos nutricionales libres de tirosina y fenilalanina. Además, su uso cuenta con plausibilidad biológica, lo cual va en línea con lo recomendado por el experto clínico. Por todo lo mencionado, el Instituto de Evaluaciones de Tecnologías en Salud e Investigación ­ IETSI, aprueba el uso de suplementos libres de tirosina y fenilalanina para el manejo de pacientes menores de 18 años, con diagnóstico de HT-1, según lo establecido en el Anexo N° 1 La vigencia del presente dictamen preliminar es de dos años a partir de la fecha de publicación. Así, la continuación de dicha aprobación estará sujeta a la evaluación de los resultados obtenidos y de nueva evidencia que pueda surgir en el tiempo.

Importance of amino acids Leu135 and Tyr236 for the interaction between EhCFIm25 and RNA: a molecular dynamics simulation study.

The CFIm25 subunit of the heterotetrameric cleavage factor Im (CFIm) is a critical factor in the formation of the poly(A) tail at mRNA 3' end, regulating the recruitment of polyadenylation factors, poly(A) site selection, and cleavage/polyadenylation reactions. We previously reported the homologous protein (EhCFIm25) in Entamoeba histolytica, the protozoan causing human amoebiasis, and showed the relevance of conserved Leu135 and Tyr236 residues for RNA binding. We also identified the GUUG sequence as the recognition site of EhCFIm25. To understand the interactions network that allows the EhCFIm25 to maintain its three-dimensional structure and function, here we performed molecular dynamics simulations of wild-type (WT) and mutant proteins, alone or interacting with the GUUG molecule. Our results indicated that in the presence of the GUUG sequence, WT converged more quickly to lower RMSD values in comparison with mutant proteins. However, RMSF values showed that movements of amino acids of WT and EhCFIm25*L135 T were almost identical, interacting or not with the GUUG molecule. Interestingly, EhCFIm25*L135 T, which is the only mutant with a slight RNA binding activity experimentally, presents the same stabilization of bend structures and alpha helices as WT, notably in the C-terminus. Moreover, WT and EhCFIm25*L135 T presented almost the same number of contacts that mainly involve lysine residues interacting with the G4 nucleotide. Overall, our data proposed a clear description of the structural and mechanistic data that govern the RNA binding capacity of EhCFIm25.

Aggregation of α- and ß- caseins induced by peroxyl radicals involves secondary reactions of carbonyl compounds as well as di-tyrosine and di-tryptophan formation.

The present work examined the role of Tyr and Trp in oxidative modifications of caseins, the most abundant milk proteins, induced by peroxyl radicals (ROO•). We hypothesized that the selectivity of ROO• and the high flexibility of caseins (implying a high exposure of Tyr and Trp residues) would favor radical-radical reactions, and di-tyrosine (di-Tyr) and di-tryptophan (di-Trp) formation. Solutions of α- and ß-caseins were exposed to ROO• from thermolysis and photolysis of AAPH (2,2'-azobis(2-methylpropionamidine)dihydrochloride). Oxidative modifications were examined using electrophoresis, western blotting, fluorescence, and chromatographic methodologies with diode array, fluorescence and mass detection. Exposure of caseins to AAPH at 37 °C gave fragmentation, cross-linking and protein aggregation. Amino acid analysis showed consumption of Trp, Tyr, Met, His and Lys residues. Quantification of Trp and Tyr products, showed low levels of di-Tyr and di-Trp, together with an accumulation of carbonyls indicating that casein aggregation is, at least partly, associated with secondary reactions between carbonyls and Lys and His residues. AAPH photolysis, which generates a high flux of free radicals increased the extent of formation of di-Tyr in both model peptides and α- and ß- caseins; di-Trp was only detected in peptides and α-casein. Thus, in spite of the high flexibility of caseins, which would be expected to favor radical-radical reactions, the low flux of ROO• generated during AAPH thermolysis disfavours the formation of dimeric radical-radical cross-links such as di-Tyr and di-Trp, instead favoring other O2-dependent crosslinking pathways such as those involving secondary reactions of initial carbonyl products.