Comparison of cysteine content in whole proteomes across the three domains of life.

An empirical observation suggests that Giardia lamblia proteins have larger cysteine content than their counterparts in other organisms. As this parasite lacks conventional antioxidant stress systems, it is generally accepted that high cysteine content helps G. lamblia cope with oxygen toxicity, a strategy apparently shared by other organisms. Here, we question whether the high cysteine content in some organisms is genuine or just a simple assumption based on singular observations. To this end, we analyzed the cysteine content in 78 proteomes of organisms spanning the three domains of life. The results indicate that the cysteine content in eukaryota is approximately double that in archaea and bacteria, with G. lamblia among the highest. Atypical cysteine contents were found in a few organisms correlating with specific environmental conditions, supporting the evolutionary amino acid-level selection of amino acid composition.

Low Expression of BRCA1 as a Potential Relapse Predictor in B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) is the most common childhood hematological malignancy worldwide. Treatment outcomes have improved dramatically in recent years; despite this, relapse is still a problem, and the potential molecular explanation for this remains an important field of study. We performed microarray and single-cell RNA-Seq data mining, and we selected significant data with a P -value<0.05. We validated BRCA1 gene expression by means of quantitative (reverse transcription-polymerase chain reaction.) We performed statistical analysis and considered a P -value<0.05 significant. We identified the overexpression of breast cancer 1, early onset (BRCA1; P -value=2.52 -134 ), by means of microarray analysis. Moreover, the normal distribution of BRCA1 expression in healthy bone marrow. In addition, we confirmed the increases in BRCA1 expression using real-time (reverse transcription-polymerase chain reaction and determined that it was significantly reduced in patients with relapse ( P -values=0.026). Finally, we identified that the expression of the BRCA1 gene could predict early relapse ( P -values=0.01). We determined that low expression of BRCA1 was associated with B-cell acute lymphoblastic leukemia relapse and could be a potential molecular prognostic marker.

Immunoproteomics of cow's milk allergy in Mexican pediatric patients.

Immunological mechanisms of non-IgE-mediated cow's milk protein allergy (CMPA) are not well understood. Such a circumstance requires attention with the aim of discovering new biomarkers that could lead to better diagnostic assays for early treatment. Here, we sought both to investigate the mechanism that underlies non-IgE-mediated CMPA and to identify cow's milk immunoreactive proteins in a Mexican pediatric patient group (n = 34). Hence, we determined the IgE and IgG1-4 subclass antibody levels against cow's milk proteins (CMP) by ELISA. Then, we performed 2D-Immunoblots using as first antibody immunoglobulins in the patients'serum that bound specifically against CMP together with CMP enrichment by ion-exchange chromatography. Immunoreactive proteins were identified by mass spectrometry-based proteomics. The serological test confirmed absence of specific IgE in the CMPA patients but showed significant increase in antigen-specific IgG1. Additionally, we identified 11 proteins that specifically bound to IgG1. We conclude that the detection of specific IgG1 together with an immunoproteomics approach is highly relevant to the understanding of CMPA's physiopathology and as a possible aid in making a prognosis since current evidence indicates IgG1 occurrence as an early signal of potential risk toward development of IgE-mediated food allergy. SIGNIFICANCE: Allergies are one of the most studied topics in the field of public health and novel protein allergens are found each year. Discovery of new principal and regional allergens has remarkable repercussions in precise molecular diagnostics, prognostics, and more specific immunotherapies. In this context, specific IgE is widely known to mediate physiopathology; however, allergies whose mechanism does not involve this immunoglobulin are poorly understood although their incidence has increased. Therefore, accurate diagnosis and adequate treatment are delayed with significant consequences on the health of pediatric patients. The study of type and subtypes of immunoglobulins associated with the immunoreactivity of cow's milk proteins together with an immunoproteomics approach allows better comprehension of physiopathology, brings the opportunity to discover new potential cow's milk protein allergens and may help in prognosis prediction (IgG1 occurrence as an early signal of possible risk toward development of IgE-mediated food allergy).

Finding the First Potential Inhibitors of Shikimate Kinase from Methicillin Resistant Staphylococcus aureus through Computer-Assisted Drug Design.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important threat as it causes serious hospital and community acquired infections with deathly outcomes oftentimes, therefore, development of new treatments against this bacterium is a priority. Shikimate kinase, an enzyme in the shikimate pathway, is considered a good target for developing antimicrobial drugs; this is given because of its pathway, which is essential in bacteria whereas it is absent in mammals. In this work, a computer-assisted drug design strategy was used to report the first potentials inhibitors for Shikimate kinase from methicillin-resistant Staphylococcus aureus (SaSK), employing approximately 5 million compounds from ZINC15 database. Diverse filtering criteria, related to druglike characteristics and virtual docking screening in the shikimate binding site, were performed to select structurally diverse potential inhibitors from SaSK. Molecular dynamics simulations were performed to elucidate the dynamic behavior of each SaSK-ligand complex. The potential inhibitors formed important interactions with residues that are crucial for enzyme catalysis, such as Asp37, Arg61, Gly82, and Arg138. Therefore, the compounds reported provide valuable information and can be seen as the first step toward developing SaSK inhibitors in the search of new drugs against MRSA.

Design, synthesis, kinetic, molecular dynamics, and hypoglycemic effect characterization of new and potential selective benzimidazole derivatives as Protein Tyrosine Phosphatase 1B inhibitors.

Protein-tyrosine phosphatase 1B (PTP1B) is a negative regulator of insulin signaling pathway and has been validated as a therapeutic target for type 2 diabetes. A wide variety of scaffolds have been included in the structure of PTP1B inhibitors, one of them is the benzimidazole nucleus. Here, we report the design and synthesis of a new series of di- and tri- substituted benzimidazole derivatives including their kinetic and structural characterization as PTP1B inhibitors and hypoglycemic activity. Results show that compounds 43, 44, 45, and 46 are complete mixed type inhibitors with a Ki of 12.6 µM for the most potent (46). SAR type analysis indicates that a chloro substituent at position 6(5), a ß-naphthyloxy at position 5(6), and a p-benzoic acid attached to the linker 2-thioacetamido at position 2 of the benzimidazole nucleus, was the best combination for PTP1B inhibition and hypoglycemic activity. In addition, molecular dynamics studies suggest that these compounds could be potential selective inhibitors from other PTPs such as its closest homologous TCPTP, SHP-1, SHP-2 and CDC25B. Therefore, the compounds reported here are good hits that provide structural, kinetic, and biological information that can be used to develop novel and selective PTP1B inhibitors based on benzimidazole scaffold.

Discovery of Benzopyrrolizidines as Promising Antigiardiasic Agents.

Current treatments for giardiasis include drugs with undesirable side effects, which increase the levels of therapeutic desertion and promote drug resistance in the parasites. Herein, we describe the antigiardiasic evaluation on Giardia lamblia trophozoites of a structurally diverse collection of 74 molecules. Among these scaffolds, we discovered a benzopyrrolizidine derivative with higher antigiardiasic activity (IC50 = 11 µM) and lower cytotoxicity in human cell cultures (IC50 = 130 µM) than those displayed by the current gold-standard drugs (metronidazole and tinidazole). Furthermore, this compound produced morphologic modifications of trophozoites, with occasional loss of one of the nuclei, among other changes not observed with standard giardicidal drugs, suggesting that it might act through a novel mechanism of action.

Structure-based identification of a potential non-catalytic binding site for rational drug design in the fructose 1,6-biphosphate aldolase from Giardia lamblia.

Giardia lamblia is the causal agent of giardiasis, one of the most prevalent parasitosis in the world. Even though effective pharmacotherapies against this parasite are available, the disadvantages associated with its use call for the development of new antigiardial compounds. Based on the Giardia dependence on glycolysis as a main energy source, glycolytic enzymes appear to be attractive targets with antiparasitic potential. Among these, fructose 1,6-biphosphate aldolase (GlFBPA) has been highlighted as a promising target for drug design. Current efforts are based on the design of competitive inhibitors of GlFBPA; however, in the kinetic context of metabolic pathways, competitive inhibitors seem to have low potential as therapeutic agents. In this work, we performed an experimental and in silico structure-based approach to propose a non-catalytic binding site which could be used as a hot spot for antigardial drug design. The druggability of the selected binding site was experimentally tested; the alteration of the selected region by site directed mutagenesis disturbs the catalytic properties and the stability of the enzyme. A computational automated search of binding sites supported the potential of this region as functionally relevant. A preliminary docking study was performed, in order to explore the feasibility and type of molecules to be able to accommodate in the proposed binding region. Altogether, the results validate the proposed region as a specific molecular binding site with pharmacological potential.

Biochemical, Kinetic, and Computational Structural Characterization of Shikimate Kinase from Methicillin-Resistant Staphylococcus aureus.

One of the most widespread pathogens worldwide is methicillin-resistant Staphylococcus aureus, a bacterium that provokes severe life-threatening illnesses both in hospitals and in the community. The principal challenge lies in the resistance of MRSA to current treatments, which encourages the study of different molecular targets that could be used to develop new drugs against this infectious agent. With this goal, a detailed characterization of shikimate kinase from this microorganism (SaSK) is described. The results showed that SaSK has a Km of 0.153 and 224 µM for shikimate and ATP, respectively, and a global reaction rate of 13.4 µmol/min/mg; it is suggested that SaSK utilizes the Bi-Bi Ping Pong reaction mechanism. Furthermore, the physicochemical data indicated that SaSK is an unstable, hydrophilic, and acidic protein. Finally, structural information showed that SaSK presented folding that is typical of its homologous counterparts and contains the typical domains of this family of proteins. Amino acids that have been shown to be important for SaSK protein function are conserved. Therefore, this study provides fundamental information that may aid in the design of inhibitors that could be used to develop new antibacterial agents.

Disulfiram as a novel inactivator of Giardia lamblia triosephosphate isomerase with antigiardial potential.

Giardiasis, the infestation of the intestinal tract by Giardia lamblia, is one of the most prevalent parasitosis worldwide. Even though effective therapies exist for it, the problems associated with its use indicate that new therapeutic options are needed. It has been shown that disulfiram eradicates trophozoites in vitro and is effective in vivo in a murine model of giardiasis; disulfiram inactivation of carbamate kinase by chemical modification of an active site cysteine has been proposed as the drug mechanism of action. The triosephosphate isomerase from G. lamblia (GlTIM) has been proposed as a plausible target for the development of novel antigiardial pharmacotherapies, and chemical modification of its cysteine 222 (C222) by thiol-reactive compounds is evidenced to inactivate the enzyme. Since disulfiram is a cysteine modifying agent and GlTIM can be inactivated by modification of C222, in this work we tested the effect of disulfiram over the recombinant and trophozoite-endogenous GlTIM. The results show that disulfiram inactivates GlTIM by modification of its C222. The inactivation is species-specific since disulfiram does not affect the human homologue enzyme. Disulfiram inactivation induces only minor conformational changes in the enzyme, but substantially decreases its stability. Recombinant and endogenous GlTIM inactivates similarly, indicating that the recombinant protein resembles the natural enzyme. Disulfiram induces loss of trophozoites viability and inactivation of intracellular GlTIM at similar rates, suggesting that both processes may be related. It is plausible that the giardicidal effect of disulfiram involves the inactivation of more than a single enzyme, thus increasing its potential for repurposing it as an antigiardial drug.

Species-Specific Inactivation of Triosephosphate Isomerase from Trypanosoma brucei: Kinetic and Molecular Dynamics Studies.

Human African Trypanosomiasis (HAT), a disease that provokes 2184 new cases a year in Sub-Saharan Africa, is caused by Trypanosoma brucei. Current treatments are limited, highly toxic, and parasite strains resistant to them are emerging. Therefore, there is an urgency to find new drugs against HAT. In this context, T. brucei depends on glycolysis as the unique source for ATP supply; therefore, the enzyme triosephosphate isomerase (TIM) is an attractive target for drug design. In the present work, three new benzimidazole derivatives were found as TbTIM inactivators (compounds 1, 2 and 3) with an I50 value of 84, 82 and 73 µM, respectively. Kinetic analyses indicated that the three molecules were selective when tested against human TIM (HsTIM) activity. Additionally, to study their binding mode in TbTIM, we performed a 100 ns molecular dynamics simulation of TbTIM-inactivator complexes. Simulations showed that the binding of compounds disturbs the structure of the protein, affecting the conformations of important domains such as loop 6 and loop 8. In addition, the physicochemical and drug-like parameters showed by the three compounds suggest a good oral absorption. In conclusion, these molecules will serve as a guide to design more potent inactivators that could be used to obtain new drugs against HAT.

Purification, concentration and recovery of small fragments of DNA from Giardia lamblia and their use for other molecular techniques.

Purification of nucleic acids is an essential procedure for most experiments in molecular biology. In this paper, the freeze-squeeze method with some modifications is proposed as an alternative methodology for the purification, concentration and recovery of small DNA fragments from agarose gels. The advantage of this alternative methodology is that it enables the recovery of fragments that are less than 100 bp in length and enables suspension of products in smaller volumes compared to several commercially available kits. In addition, the purified fragments were re-amplified by PCR and used for cloning and sequencing. Moreover, this protocol was used to perform the isolation and identification of microRNAs from Giardia lamblia, as previously reported. This protocol has the advantage of being inexpensive and easy and can be employed for various molecular applications. The advantages of this protocol include •A modified classical method was used for purification of small DNA fragments from G. lamblia.•The modified freeze-squeeze method was more efficient in cleaning up small DNA fragments from agarose gels compared to commercial kits.•The modified method allows concentration and recovery of fragments up to 60 bp in length.•The modified freeze-squeeze method allows re-suspension of the products in volumes of up to 2.5 µL.

Proteomics: a tool to develop novel diagnostic methods and unravel molecular mechanisms of pediatric diseases / Proteómica: una herramienta para desarrollar nuevos métodos de diagnóstico y descifrar mecanismos moleculares de enfermedades pediátricas

Abstract: Proteomics is the study of the expression of changes and post-translational modifications (PTM) of proteins along a metabolic condition either normal or pathological. In the field of health, proteomics allows obtaining valuable data for treatment, diagnosis or pathophysiological mechanisms of different illnesses. To illustrate the aforementioned, we describe two projects currently being performed at the Instituto Nacional de Pediatría: The immuno-proteomic study of cow milk allergy and the Proteomic study of childhood cataract. Cow's milk proteins (CMP) are the first antigens to which infants are exposed and generate allergy in some of them. In Mexico, the incidence of CMP allergy has been estimated at 5-7%. Clinical manifestations include both gastrointestinal and extra-gastrointestinal symptoms, making its diagnosis extremely difficult. An inappropriate diagnosis affects the development and growth of children. The goals of the study are to identify the main immune-reactive CMP in Mexican pediatric population and to design more accurate diagnostic tools for this disease. Childhood cataract is a major ocular disease representing one of the main causes of blindness in infants; in developing countries, this disease promotes up to 27% of cases related to visual loss. From this group, it has been estimated that close to 60% of children do not survive beyond two years after vision lost. PTM have been pointed out as the main cause of protein precipitation at the crystalline and, consequently, clouding of this tissue. The study of childhood cataract represents an outstanding opportunity to identify the PTM associated to the cataract-genesis process.

Resumen: La proteómica estudia los cambios de expresión y post-traduccionales (PTM) de las proteínas durante una condición metabólica normal o patológica. En el campo de la salud, la proteómica permite obtener datos útiles para el tratamiento, diagnóstico o en la fisiopatología de diferentes enfermedades. Para ilustrar lo anterior, describimos dos proyectos realizados en el Instituto Nacional de Pediatría: El estudio inmunoproteómico de la alergia a la leche y el estudio proteómico de la catarata infantil. Las proteínas de leche bovina (PLB) son los primeros antígenos a los que se exponen los infantes y un porcentaje de ellos generará alergias. En México, se estima que la incidencia de alergias a las PLB es del 5-7%. Las manifestaciones clínicas incluyen tanto síntomas gastrointestinales como extra-gastrointestinales, dificultando su diagnóstico. Un mal diagnóstico afecta el desarrollo y crecimiento del infante. Los objetivos del estudio son identificar las principales PLB inmunoreactivas en población infantil mexicana y diseñar herramientas diagnósticas más precisas para esta patología. La catarata infantil es una enfermedad ocular que representa una de las causas principales de ceguera infantil; en países subdesarrollados genera cerca del 27% de casos relacionados con pérdida visual. De este grupo, se estima que cerca del 60% de los infantes no sobreviven más allá de los dos años después de perder la visión. Se señala a las PTM como las responsables de la precipitación de proteínas del cristalino y, por tanto, de su opacidad. El estudio de la catarata infantil representa una oportunidad para identificar las PTM vinculadas con la cataratogénesis.

Proteomics: a tool to develop novel diagnostic methods and unravel molecular mechanisms of pediatric diseases.

Proteomics is the study of the expression of changes and post-translational modifications (PTM) of proteins along a metabolic condition either normal or pathological. In the field of health, proteomics allows obtaining valuable data for treatment, diagnosis or pathophysiological mechanisms of different illnesses. To illustrate the aforementioned, we describe two projects currently being performed at the Instituto Nacional de Pediatría: The immuno-proteomic study of cow milk allergy and the Proteomic study of childhood cataract. Cow's milk proteins (CMP) are the first antigens to which infants are exposed and generate allergy in some of them. In Mexico, the incidence of CMP allergy has been estimated at 5-7%. Clinical manifestations include both gastrointestinal and extra-gastrointestinal symptoms, making its diagnosis extremely difficult. An inappropriate diagnosis affects the development and growth of children. The goals of the study are to identify the main immune-reactive CMP in Mexican pediatric population and to design more accurate diagnostic tools for this disease. Childhood cataract is a major ocular disease representing one of the main causes of blindness in infants; in developing countries, this disease promotes up to 27% of cases related to visual loss. From this group, it has been estimated that close to 60% of children do not survive beyond two years after vision lost. PTM have been pointed out as the main cause of protein precipitation at the crystalline and, consequently, clouding of this tissue. The study of childhood cataract represents an outstanding opportunity to identify the PTM associated to the cataract-genesis process.

Stem-Loop RT-qPCR as an Efficient Tool for the Detection and Quantification of Small RNAs in Giardia lamblia.

Stem-loop quantitative reverse transcription PCR (RT-qPCR) is a molecular technique used for identification and quantification of individual small RNAs in cells. In this work, we used a Universal ProbeLibrary (UPL)-based design to detect-in a rapid, sensitive, specific, and reproducible way-the small nucleolar RNA (snoRNA) GlsR17 and its derived miRNA (miR2) of Giardia lamblia using a stem-loop RT-qPCR approach. Both small RNAs could be isolated from both total RNA and small RNA samples. Identification of the two small RNAs was carried out by sequencing the PCR-amplified small RNA products upon ligation into the pJET1.2/blunt vector. GlsR17 is constitutively expressed during the 72 h cultures of trophozoites, while the mature miR2 is present in 2-fold higher abundance during the first 48 h than at 72 h. Because it has been suggested that miRNAs in G. lamblia have an important role in the regulation of gene expression, the use of the stem-loop RT-qPCR method could be valuable for the study of miRNAs of G. lamblia. This methodology will be a powerful tool for studying gene regulation in G. lamblia, and will help to better understand the features and functions of these regulatory molecules and how they work within the RNA interference (RNAi) pathway in G. lamblia.

Cloning, Expression and Characterization of Recombinant, NADH Oxidase from Giardia lamblia.

The NADH oxidase family of enzymes catalyzes the oxidation of NADH by reducing molecular O2 to H2O2, H2O or both. In the protozoan parasite Giardia lamblia, the NADH oxidase enzyme (GlNOX) produces H2O as end product without production of H2O2. GlNOX has been implicated in the parasite metabolism, the intracellular redox regulation and the resistance to drugs currently used against giardiasis; therefore, it is an interesting protein from diverse perspectives. In this work, the GlNOX gene was amplified from genomic G. lamblia DNA and expressed in Escherichia coli as a His-Tagged protein; then, the enzyme was purified by immobilized metal affinity chromatography, characterized, and its properties compared with those of the endogenous enzyme previously isolated from trophozoites (Brown et al. in Eur J Biochem 241(1):155-161, 1996). In comparison with the trophozoite-extracted enzyme, which was scarce and unstable, the recombinant heterologous expression system and one-step purification method produce a stable protein preparation with high yield and purity. The recombinant enzyme mostly resembles the endogenous protein; where differences were found, these were attributable to methodological discrepancies or artifacts. This homogenous, pure and functional protein preparation can be used for detailed structural or functional studies of GlNOX, which will provide a deeper understanding of the biology and pathogeny of G. lamblia.

Structural effects of protein aging: terminal marking by deamidation in human triosephosphate isomerase.

Deamidation, the loss of the ammonium group of asparagine and glutamine to form aspartic and glutamic acid, is one of the most commonly occurring post-translational modifications in proteins. Since deamidation rates are encoded in the protein structure, it has been proposed that they can serve as molecular clocks for the timing of biological processes such as protein turnover, development and aging. Despite the importance of this process, there is a lack of detailed structural information explaining the effects of deamidation on the structure of proteins. Here, we studied the effects of deamidation on human triosephosphate isomerase (HsTIM), an enzyme for which deamidation of N15 and N71 has been long recognized as the signal for terminal marking of the protein. Deamidation was mimicked by site directed mutagenesis; thus, three mutants of HsTIM (N15D, N71D and N15D/N71D) were characterized. The results show that the N71D mutant resembles, structurally and functionally, the wild type enzyme. In contrast, the N15D mutant displays all the detrimental effects related to deamidation. The N15D/N71D mutant shows only minor additional effects when compared with the N15D mutation, supporting that deamidation of N71 induces negligible effects. The crystal structures show that, in contrast to the N71D mutant, where minimal alterations are observed, the N15D mutation forms new interactions that perturb the structure of loop 1 and loop 3, both critical components of the catalytic site and the interface of HsTIM. Based on a phylogenetic analysis of TIM sequences, we propose the conservation of this mechanism for mammalian TIMs.

Deficiencia de glucosa-6-fosfatodeshidrogenasa: De lo clínico a lo bioquímico / Glucose-6-phosphate dehydrogenase: From the clinical to the biochemical aspects / Deficiência de glicose-6-fosfato desidrogenase: Aspectos clínicos e bioquímicos

La deficiencia de Glucosa-6-fosfato deshidrogenasa (G6PD) es la enzimopatíamás frecuente, con una prevalencia global del 4,9% y con alrededor de 330 a 400 millones de personas afectadas en el mundo. La G6PD desempeña un papel fundamental en el equilibrio redox intracelular, especialmente en los eritrocitos; en condiciones de estrés oxidativo inducido (por ejemplo,por exposición a agentes externos como fármacos, alimentos o infecciones),los hematíes portadores de la variante enzimática y con deficiencia de la actividad enzimática, sufren daños irreversibles que condicionan su destrucción acelerada. La hemólisis explica el espectro de manifestaciones clínicas de esta enfermedad, que incluyen ictericia neonatal, episodios de hemólisis aguda inducida por agentes externos o anemia hemolítica crónica. El presente trabajo hace una reseña de los aspectos epidemiológicos y clínicos de esta enfermedad y revisa los aspectos fisiopatológicos a nivel bioquímico-molecular, con particular énfasis en la caracterización genética,estructural y funcional de las variantes asociadas a la deficiencia de G6PD.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most frequent enzymopathy in humans with a global prevalence of 4.9 % and around 330 to 400 million patients affected worldwide. G6PD plays a fundamental role in the intracellular redox equilibrium, especially in red blood cells (RBC). Under oxidative stress (induced by exposure to external agents like drugs, infections or diet) RBC carrying the deficient variant suffer irreversible damage resulting in their accelerated destruction. This hemolysis explains the clinical manifestations of the disease that include neonatal jaundice, inducedacute hemolysis or chronic hemolytic anemia. This work summarizes the epidemiologic and clinical features of G6PD deficiency, and reviews the molecular pathophysiology of this disease with special emphasis on the genetical, structural and functional characterization of variants causing this pathology.

A deficiência da Glicose-6-FosFato desidrogenase (G6PD) é a enzimopatia mais Frequente, com uma prevalência global do 4,9%, e com aproximadamente 330 a 400 milhões de pessoas afetadas no mundo. A G6PD tem um importante papel no equilíbrio celular redox intracelular, especialmente nos eritrócitos; em condições de estresse oxidativo induzido, (por exemplo, pela exposição a agentes externos como Fármacos, alimentos, ou infecções) as hemácias portadoras da variante enzimática e com defciência da atividade enzimática, sofrem danos irreversíveis que condicionam a sua destruição acelerada. A hemólise explica o espectro de manifestações clínicas desta doença, que incluem icterícia neonatal, episódios de hemólise aguda induzida por agentes externos ou anemia hemolítica crônica. Este trabalho faz uma resenha dos aspectos epidemiológicos e clínicos desta doença, e revisa os aspectos fsiopatológicos no nível bioquímico-molecular, com ênfase especial na caracterização genética, estrutural e funcional das variantes associadas à defciência de G6PD.

Giardial triosephosphate isomerase as possible target of the cytotoxic effect of omeprazole in Giardia lamblia.

Giardiasis is highly prevalent in the developing world, and treatment failures with the standard drugs are common. This work deals with the proposal of omeprazole as a novel antigiardial drug, focusing on a giardial glycolytic enzyme used to follow the cytotoxic effect at the molecular level. We used recombinant technology and enzyme inactivation to demonstrate the capacity of omeprazole to inactivate giardial triosephosphate isomerase, with no adverse effects on its human counterpart. To establish the specific target in the enzyme, we used single mutants of every cysteine residue in triosephosphate isomerase. The effect on cellular triosephosphate isomerase was evaluated by following the remnant enzyme activity on trophozoites treated with omeprazole. The interaction of omeprazole with giardial proteins was analyzed by fluorescence spectroscopy. The susceptibility to omeprazole of drug-susceptible and drug-resistant strains of Giardia lamblia was evaluated to demonstrate its potential as a novel antigiardial drug. Our results demonstrate that omeprazole inhibits giardial triosephosphate isomerase in a species-specific manner through interaction with cysteine at position 222. Omeprazole enters the cytoplasmic compartment of the trophozoites and inhibits cellular triosephosphate isomerase activity in a dose-dependent manner. Such inhibition takes place concomitantly with the cytotoxic effect caused by omeprazole on trophozoites. G. lamblia triosephosphate isomerase (GlTIM) is a cytoplasmic protein which can help analyses of how omeprazole works against the proteins of this parasite and in the effort to understand its mechanism of cytotoxicity. Our results demonstrate the mechanism of giardial triosephosphate isomerase inhibition by omeprazole and show that this drug is effective in vitro against drug-resistant and drug-susceptible strains of G. lamblia.

The E104D mutation increases the susceptibility of human triosephosphate isomerase to proteolysis. Asymmetric cleavage of the two monomers of the homodimeric enzyme.

The deficiency of human triosephosphate isomerase (HsTIM) generates neurological alterations, cardiomyopathy and premature death. The mutation E104D is the most frequent cause of the disease. Although the wild type and mutant exhibit similar kinetic parameters, it has been shown that the E104D substitution induces perturbation of an interfacial water network that, in turn, reduces the association constant between subunits promoting enzyme inactivation. To gain further insight into the effects of the mutation on the structure, stability and function of the enzyme, we measured the sensitivity of recombinant E104D mutant and wild type HsTIM to limited proteolysis. The mutation increases the susceptibility to proteolysis as consequence of the loss of rigidity of its overall 3-D structure. Unexpectedly, it was observed that proteolysis of wild type HsTIM generated two different stable nicked dimers. One was formed in relatively short times of incubation with proteinase K; as shown by spectrometric and crystallographic data, it corresponded to a dimer containing a nicked monomer and an intact monomer. The formation of the other nicked species requires relatively long incubation times with proteinase K and corresponds to a dimer with two clipped subunits. The first species retains 50% of the original activity, whereas the second species is inactive. Collectively, we found that the E104D mutant is highly susceptible to proteolysis, which in all likelihood contributes to the pathogenesis of enzymopathy. In addition, the proteolysis data on wild type HsTIM illustrate an asymmetric conduct of the two monomers.