How to Design U1 snRNA Molecules for Splicing Rescue.

Mutations affecting constitutive splice donor sites (5'ss) are among the most frequent genetic defects that disrupt the normal splicing process. Pre-mRNA splicing requires the correct identification of a number of cis-acting elements in an ordered fashion. By disrupting the complementarity of the 5'ss with the endogenous small nuclear RNA U1 (U1 snRNA), the key component of the spliceosomal U1 ribonucleoprotein, 5'ss mutations may result in exon skipping, intron retention or activation of cryptic splice sites. Engineered modification of the U1 snRNA seemed to be a logical method to overcome the effect of those mutations. In fact, over the last years, a number of in vitro studies on the use of those modified U1 snRNAs to correct a variety of splicing defects have demonstrated the feasibility of this approach. Furthermore, recent reports on its applicability in vivo are adding up to the principle that engineered modification of U1 snRNAs represents a valuable approach and prompting further studies to demonstrate the clinical translatability of this strategy.Here, we outline the design and generation of U1 snRNAs with different degrees of complementarity to mutated 5'ss. Using the HGSNAT gene as an example, we describe the methods for a proper evaluation of their efficacy in vitro, taking advantage of our experience to share a number of tips on how to design U1 snRNA molecules for splicing rescue.

Development of an Antisense Oligonucleotide-Mediated Exon Skipping Therapeutic Strategy for Mucolipidosis II: Validation at RNA Level.

Lysosomal storage disorders (LSDs) are a group of rare inherited metabolic diseases caused by the malfunction of the lysosomal system, which results in the accumulation of undergraded substrates inside the lysosomes and leads to severe and progressive pathology. Despite there currently being a broad understanding of the molecular defects behind LSDs, curative therapies have been approved for only few of these diseases, whereas existing treatments are still mostly symptomatic with several limitations. Mucolipidosis type II alpha/beta (ML II) is one of most severe LSDs, which is caused by the total deficiency of the GlcNAc-1-phosphotransferase, a key enzyme for the formation of specific targeting signals on lysosomal hydrolases to lysosomes. GlcNAc-1-phosphotransferase is a multimeric enzyme complex encoded by two genes: GNPTAB and GNPTG. One of the most frequent ML II causal mutation is a dinucleotide deletion on exon 19 of GNPTAB (c.3503_3504del) that leads to the generation of a truncated protein, loss of GlcNAc-1-phosphotransferase activity, and missorting of multiple lysosomal enzymes. Presently, there is no therapy available for ML II. In this study, we explored the possibility of an innovative therapeutic strategy for ML II based on the use of antisense oligonucleotides (AOs) capable to induce the skipping of GNPTAB exon 19 harboring the most common disease-causing mutation, c.3503_3504del. The approach confirmed the ability of specific AOs for RNA splicing modulation, thus paving the way for future studies on the therapeutic potential of this strategy.

Structural and functional characterization of suramin-bound MjTX-I from Bothrops moojeni suggests a particular myotoxic mechanism.

Local myonecrosis is the main event resulting from snakebite envenomation by the Bothrops genus and, frequently, it is not efficiently neutralized by antivenom administration. Proteases, phospholipases A2 (PLA2) and PLA2-like toxins are found in venom related to muscle damage. Functional sites responsible for PLA2-like toxins activity have been proposed recently; they consist of a membrane docking-site and a membrane rupture-site. Herein, a combination of functional, biophysical and crystallographic techniques was used to characterize the interaction between suramin and MjTX-I (a PLA2-like toxin from Bothrops moojeni venom). Functional in vitro neuromuscular assays were performed to study the biological effects of the protein-ligand interaction, demonstrating that suramin neutralizes the myotoxic effect of MjTX-I. Calorimetric assays showed two different binding events: (i) inhibitor-protein interactions and (ii) toxin oligomerization processes. These hypotheses were also corroborated with dynamic light and small angle X-ray scattering assays. The crystal structure of the MjTX-I/suramin showed a totally different interaction mode compared to other PLA2-like/suramin complexes. Thus, we suggested a novel myotoxic mechanism for MjTX-I that may be inhibited by suramin. These results can further contribute to the search for inhibitors that will efficiently counteract local myonecrosis in order to be used as an adjuvant of conventional serum therapy.

Biophysical study of human induced Pluripotent Stem Cell-Derived cardiomyocyte structural maturation during long-term culture.

Human induced Pluripotent Stem Cell-derived cardiomyocytes (hiPSC-CMs) have an enormous potential for the development of drug screening and modeling cardiac disease platforms. However, early hiPSC-CMs usually exhibit low structural development, precluding the applicability of these cells. Here, we follow during 120 days the progressive structural maturation of hiPSC-CM microtissues obtained using the Wnt signaling modulation protocol. For this purpose, we designed a user friendly custom-written program to quantify cardiac fiber alignment and sarcomere length. Cardiomyocyte shape, cardiac fiber density and multinucleation were also analyzed. Derived cardiomyocytes showed significant progression in cardiomyocyte fiber density and sarcomere length during the long-term culture, with a peak at day 90 of 40% multinucleated cells. In addition, cardiomyocyte microtissues remained functional with progressive maturation leading to a decrease in the percentage of cTnT positive cells from 59% to 22% at day 120, a value similar to the content present in tissues of the adult left ventricle. These data and the framework that we provide to quantify cardiomyocyte structural features can be important to set new metrics to develop applications for drug screening and disease modeling.

Structural evidence for a fatty acid-independent myotoxic mechanism for a phospholipase A2-like toxin.

The myotoxic mechanism for PLA2-like toxins has been proposed recently to be initiated by an allosteric change induced by a fatty acid binding to the protein, leading to the alignment of the membrane docking site (MDoS) and membrane disrupting site (MDiS). Previous structural studies performed by us demonstrated that MjTX-II, a PLA2-like toxin isolated from Bothrops moojeni, presents a different mode of ligand-interaction caused by natural amino acid substitutions and an insertion. Herein, we present four crystal structures of MjTX-II, in its apo state and complexed with fatty acids of different lengths. Analyses of these structures revealed slightly different oligomeric conformations but with both MDoSs in an arrangement that resembles an active-state PLA2-like structure. To explore the structural transitions between apo protein and fatty-acid complexes, we performed Normal Mode Molecular Dynamics simulations, revealing that oligomeric conformations of MjTX-II/fatty acid complexes may be reached in solution by the apo structure. Similar simulations with typical PLA2-like structures demonstrated that this transition is not possible without the presence of fatty acids. Thus, we hypothesize that MjTX-II does not require fatty acids to be active, although these ligands may eventually help in its stabilization by the formation of hydrogen bonds. Therefore, these results complement previous findings for MjTX-II and help us understand its particular ligand-binding properties and, more importantly, its particular mechanism of action, with a possible impact on the design of structure-based inhibitors for PLA2-like toxins in general.

Crystal structure of a phospholipase A2 from Bothrops asper venom: Insights into a new putative "myotoxic cluster".

Snake venoms from the Viperidae and Elapidae families often have several phospholipases A2 (PLA2s), which may display different functions despite having a similar structural scaffold. These proteins are considered an important target for the development of drugs against local myotoxic damage because they are not efficiently neutralized by conventional serum therapy. PLA2s from these venoms are generally divided into two classes: (i) catalytic PLA2s (or Asp49-PLA2s) and (ii) non-catalytic PLA2-like toxins (or Lys49-PLA2s). In many Viperidae venoms, a subset of the basic Asp49-PLA2s displays some functional and structural characteristics of PLA2-like proteins and group within the same phylogenetic clade, but their myotoxic mechanism is still largely unknown. In the present study, we have crystallized and solved the structure of myotoxin I (MT-I), a basic myotoxic Asp49-PLA2 isolated from Bothrops asper venom. The structure presents a dimeric conformation that is compatible with that of previous dimers found for basic myotoxic Asp49-PLA2s and Lys49-PLA2s and has been confirmed by other biophysical and bioinformatics techniques. This arrangement suggests a possible cooperative action between both monomers to exert myotoxicity via two different sites forming a putative membrane-docking site (MDoS) and a putative membrane disruption site (MDiS). This mechanism would resemble that proposed for Lys49-PLA2s, but the sites involved appear to be situated in a different region. Thus, as both sites are close to one another, they form a "myotoxic cluster", which is also found in two other basic myotoxic Asp49-PLA2s from Viperidae venoms. Such arrangement may represent a novel structural strategy for the mechanism of muscle damage exerted by the group of basic, Asp49-PLA2s found in viperid snake venoms.

Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin.

Local myonecrosis resulting from snakebite envenomation is not efficiently neutralized by regular antivenom administration. This limitation is considered to be a significant health problem by the World Health Organization. Phospholipase A2-like (PLA2-like) proteins are among the most important proteins related to the muscle damage resulting from several snake venoms. However, despite their conserved tertiary structure compared with PLA2s, their biological mechanism remains incompletely understood. Different oligomeric conformations and binding sites have been identified or proposed, leading to contradictory data in the literature. In the last few years, a comprehensive hypothesis has been proposed based on fatty-acid binding, allosteric changes and the presence of two different interaction sites. In the present study, a combination of techniques were used to fully understand the structural-functional characteristics of the interaction between suramin and MjTX-II (a PLA2-like toxin). In vitro neuromuscular studies were performed to characterize the biological effects of the protein-ligand interaction and demonstrated that suramin neutralizes the myotoxic activity of MjTX-II. The high-resolution structure of the complex identified the toxin-ligand interaction sites. Calorimetric assays showed two different binding events between the protein and the inhibitor. It is demonstrated for the first time that the inhibitor binds to the surface of the toxin, obstructing the sites involved in membrane docking and disruption according to the proposed myotoxic mechanism. Furthermore, higher-order oligomeric formation by interaction with interfacial suramins was observed, which may also aid the inhibitory process. These results further substantiate the current myotoxic mechanism and shed light on the search for efficient inhibitors of the local myonecrosis phenomenon.

Influence of peripheral whole-blood microRNA-7 and microRNA-221 high expression levels on the acquisition of castration-resistant prostate cancer: evidences from in vitro and in vivo studies.

Prostate cancer (PC) is the more frequently diagnosed neoplasia in men in developed countries. The evolution of PC to castration-resistant prostate cancer (CRPC) represents real problems of clinical management, in consequence to the limited therapeutic options. MicroRNAs (miRNAs) are small noncoding RNAs that play an important role in gene expression and function regulation. The increased evidence that miRNAs are involved in cancer development and progression has made them potential biomarkers for cancer diagnosis, prognosis, and aggressiveness. Our purpose was to identify a miRNA expression profile associated with the development of CRPC. We firstly observed a miRNA expression profile differentially expressed between the castration-resistant (CR) PC3 cell line and the hormone-sensitive LnCaP cell line, where miR-7, miR-221, and miR-222 were upregulated in PC3 (11.3-fold increase, P = 0.012; 11.3-fold increase, P = 0.002; 8.6-fold increase, P = 0.002, respectively). We also observed that the trend of miR-1233 expression levels was higher in PC3 (3.7-fold increase, P = 0.057). These miRNAs differentially expressed in vitro were studied in a peripheral whole-blood samples from PC patients. We observed that patients presenting an early CR acquisition (≤ 20 months) had higher expression levels of miR-7 and miR-221 (P = 0.034 and P = 0.036, respectively). Furthermore, we found that patients diagnosed with high-Gleason score tumors and presenting simultaneous higher miR-7 expression levels have a significant reduce time to CR compared with patients who present lower miR-7 expression levels (11 vs. 51 months, log-rank test P = 0.004). We also found that patients diagnosed with high-Gleason score tumors and higher expression levels of miR-221 have an early CRPC compared to patients with lower miR-221 expression levels (10 vs. 46 months, log-rank test P = 0.012). We observed a significantly lower overall survival in patients with higher peripheral whole-blood expression levels of miR-7 (28 vs. 116 months, log-rank test P = 0.001). Our results suggest that miR-7 and miR-221 peripheral whole-blood expression levels can be potential predictive biomarkers of CRPC development.

Higher circulating expression levels of miR-221 associated with poor overall survival in renal cell carcinoma patients.

The mechanisms involved in renal cell carcinoma (RCC) development and progression remain unclear, and new biomarkers for early detection, follow-up of the disease and prognosis are needed in routine practice to improve the diagnostic and/or prognostic accuracy. There is increasing evidence that microRNAs (miRNAs) are involved in cancer development and progression. The up-regulation of miR-221/222 has been described in several human cancers, and during RCC development, this up-regulation can modulate the metastatic process. Our purpose was to investigate the circulating expression levels of miR-221/222 as potential biomarkers for RCC detection and their influence in patients' overall survival. The circulating miR-221/222 was studied by relative quantification in 77 plasma samples. A follow-up study was undertaken to evaluate the overall survival. We observed that RCC patients presented higher circulating expression levels of miR-221 and miR-222 than healthy individuals (2(-ΔΔCt) = 2.8, P = 0.028; 2(-ΔΔCt) = 2.2, P = 0.044, respectively). The RCC patients with metastasis at diagnosis also presented higher circulating expression levels of miR-221 than patients with no metastasis (2(-ΔΔCt) = 10.9, P = 0.001). We also observed a significantly lower overall survival in patients with higher expression levels of miR-221 (48 vs 116 months, respectively; P = 0.024). Furthermore, multivariate Cox regression analysis using the tumour, nodes and metastasis stage (TNM stage); Fuhrman nuclear grade and age (≥60 years) as covariants demonstrated a higher risk of specific death by cancer in patients who presented higher expression levels of miR-221 (hazard ratio (HR) = 10.7, 95% confidence interval 1.33-85.65, P = 0.026). The concordance (c) index showed that the definition of profiles that contain information regarding tumour characteristics associated with circulating miR-221 expression information presents an increased capacity to predict the risk of death by RCC (c index model 1, 0.800 vs model 2, 0.961). Our results, which identified the plasma miR-221/222 at variable levels during RCC development, suggest that these miRNAs may have a potential as noninvasive biomarkers of RCC development.

Combined Influence of EGF+61G>A and TGFB+869T>C Functional Polymorphisms in Renal Cell Carcinoma Progression and Overall Survival: The Link to Plasma Circulating MiR-7 and MiR-221/222 Expression.

The epidermal growth factor (EGF) is responsible for the activation of intracellular signal transducers that act on cell-cycle progression, cell motility, angiogenesis and inhibition of apoptosis. However, cells can block these effects activating opposite signaling pathways, such as the transforming growth factor beta 1 (TGFß1) pathway. Thus changes in expression levels of EGF and TGFB1 in renal cells might modulate the renal cell carcinoma (RCC) development, in consequence of changes in regulatory elements of signaling networks such as the microRNAs (miRNAs). Our purpose was to investigate the synergic role of EGF+61G>A and TGFB1+869T>C polymorphisms in RCC development. Genetic polymorphisms were studied by allelic discrimination using real-time PCR in 133 RCC patients vs. 443 healthy individuals. The circulating EGF/EGFR-MAPK-related miR-7, miR-221 and miR-222 expression was analyzed by a quantitative real-time PCR in plasma from 22 RCC patients vs. 27 healthy individuals. The intermediate/high genetic proliferation profile patients carriers present a significantly reduced time-to-progression and a higher risk of an early relapse compared with the low genetic proliferation profile carriers (HR = 8.8, P = 0.038) with impact in a lower overall survival (Log rank test, P = 0.047). The RCC patients presented higher circulating expression levels of miR-7 than healthy individuals (6.1-fold increase, P<0.001). Moreover, the intermediate/high genetic proliferation profile carriers present an increase in expression levels of miR-7, miR-221 and miR-222 during the RCC development and this increase is not observed in low genetic proliferation profile (P<0.001, P = 0.004, P<0.001, respectively). The stimulus to angiogenesis, cell-cycle progression and tumoral cells invasion, through activation of EGFR/MAPK signaling pathway in intermediate/high proliferation profile carriers is associated with an early disease progression, resulting in a poor overall survival. We also demonstrated that the intermediate/high proliferation profile is an unfavorable prognostic factor of RCC and miR-7, miR-221 and miR-222 expressions may be useful phenotype biomarkers of EGFR/MAPK activation.

Renal cell carcinoma development and miRNAs: a possible link to the EGFR pathway.

Renal cell carcinoma (RCC) is the most common solid cancer of the adult kidney and the majority of RCC cases are detected accidentally. This reality and the nonexistence of a standard screening test contribute to the fact that one third of patients are diagnosed with local invasive disease or metastatic disease. miRNAs are a family of small ncRNAs that regulate gene expression and have been identified as key regulators in many biological processes including cell development, differentiation, apoptosis and proliferation. The EGF receptor signaling pathway is usually deregulated in cancer and it is suggested to have an important role in RCC. Further studies are needed to characterize deregulation of this pathway during RCC development. In this review we highlight some potential miRNAs that could be involved in the modulation of the EGF receptor pathway and consequently in RCC development.

Improvement of a predictive model of castration-resistant prostate cancer: functional genetic variants in TGFß1 signaling pathway modulation.

Prostate cancer (PC) is the most frequently diagnosed cancer in men. The acquisition of castration-resistant (CR) phenotype is associated with the activation of signaling pathways mediated by growth factors. The TGFß1 and its receptors have an important role in tumor progression, being the pro-apoptotic function modulated by the expression of TGFBR2. A single nucleotide polymorphism -875 G > A in TGFBR2 gene has been described, which may influence the expression levels of the receptor. Our purpose was to investigate the potential role of TGFBR2-875G>A in PC risk and in the response to androgen deprivation therapy (ADT). TGFBR2-875G>A polymorphism was studied by allelic discrimination using real-time polymerase chain reaction (PCR) in 891 patients with PC and 874 controls. A follow-up study was undertaken to evaluate response to ADT. The TGFBR2 and SMAD7 mRNA expression were analyzed by a quantitative real-time PCR. We found that TGFBR2-875GG homozygous patients present lower expression levels of TGFBR2 mRNA (AA/AG: 2(-ΔΔCT) =1.5, P=0.016). GG genotype was also associated with higher Gleason grade (OR=1.51, P=0.019) and increased risk of an early relapse after ADT (HR=1.47, P=0.024). The concordance (c) index analysis showed that the definition of profiles that contains information regarding tumor characteristics associated with genetic information present an increased capacity to predict the risk for CR development (c-index model 1: 0.683 vs model 2: 0.736 vs model 3: 0.746 vs model 4: 0.759). The TGFBR2-875G>A contribution to an early relapse in ADT patients, due to changes in mRNA expression, supports the involvement of TGFß1 pathway in CRPC. Furthermore, according to our results, we hypothesize the potential benefits of the association of genetic information in predictive models of CR development.

Structural and functional studies with mytoxin II from Bothrops moojeni reveal remarkable similarities and differences compared to other catalytically inactive phospholipases A2-like.

Lys49-phospholipases A2 (Lys49-PLA2s) are proteins found in bothropic snake venoms (Viperidae family) and belong to a class of proteins which presents a phospholipase A2 scaffold but are catalytically inactive. These proteins (also known as PLA2s-like toxins) exert a pronounced local myotoxic effect and are not neutralized by antivenom, being their study relevant in terms of medical and scientific interest. Despite of the several studies reported in the literature for this class of proteins only a partial consensus has been achieved concerning their functional-structural relationships. In this work, we present a comprehensive structural and functional study with the MjTX-II, a dimeric Lys49-PLA2 from Bothrops moojeni venom which includes: (i) high-resolution crystal structure; (ii) dynamic light scattering and bioinformatics studies in order to confirm its biological assembly; (iii) myographic and electrophysiological studies and, (iv) comparative studies with other Lys49-PLA2s. These comparative analyses let us to get important insights into the role of Lys122 amino acid, previously indicated as responsible for Lys49-PLA2s catalytic inactivity and added important elements to establish the correct biological assembly for this class of proteins. Furthermore, we show two unique sequential features of MjTX-II (an amino acid insertion and a mutation) in comparison to all bothropic Lys49-PLA2s that lead to a distinct way of ligand binding at the toxin's hydrophobic channel and also, allowed the presence of an additional ligand molecule in this region. These facts suggest a possible particular mode of binding for long-chain ligands that interacts with MjTX-II hydrophobic channel, a feature that may directly affect the design of structure-based ligands for Lys49-PLA2s.

Structural and functional studies of a bothropic myotoxin complexed to rosmarinic acid: new insights into Lys49-PLA2 inhibition.

Snakebite envenoming is an important public health problem in many tropical and subtropical countries, and is considered a neglected tropical disease by the World Health Organization. Most severe cases are inflicted by species of the families Elapidae and Viperidae, and lead to a number of systemic and local effects in the victim. One of the main problems regarding viperidic accidents is prominent local tissue damage whose pathogenesis is complex and involves the combined actions of a variety of venom components. Phospholipases A2 (PLA2s) are the most abundant muscle-damaging components of these venoms. Herein, we report functional and structural studies of PrTX-I, a Lys49-PLA2 from Bothops pirajai snake venom, and the influence of rosmarinic acid (RA) upon this toxin's activities. RA is a known active component of some plant extracts and has been reported as presenting anti-myotoxic properties related to bothopic envenomation. The myotoxic activity of Lys49-PLA2s is well established in the literature and although no in vivo neurotoxicity has been observed among these toxins, in vitro neuromuscular blockade has been reported for some of these proteins. Our in vitro studies show that RA drastically reduces both the muscle damage and the neuromuscular blockade exerted by PrTX-I on mice neuromuscular preparations (by ∼80% and ∼90%, respectively). These results support the hypothesis that the two effects are closely related and lead us to suggest that they are consequences of the muscle membrane-destabilizing activity of the Lys49-PLA2. Although the C-terminal region of these proteins has been reported to comprise the myotoxic site, we demonstrate by X-ray crystallographic studies that RA interacts with PrTX-I in a different region. Consequently, a new mode of Lys49-PLA2 inhibition is proposed. Comparison of our results with others in the literature suggests possible new ways to inhibit bothropic snake venom myotoxins and improve serum therapy.

Structural, functional, and bioinformatics studies reveal a new snake venom homologue phospholipase A2 class.

Phospholipases A2 (PLA2s) are enzymes responsible for membrane disruption through Ca(2+) -dependent hydrolysis of phospholipids. Lys49-PLA2s are well-characterized homologue PLA2s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA2s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA2 (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA2 conserves all the residues responsible for Ca(2+) coordination and of the catalytic network, features thought to be fundamental for PLA2 enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA2s. Crystallographic studies of the complex BthTX-II/Ca(2+) show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca(2+) are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA2s than to other Asp49-PLA2s, thus allowing Crotalinae subfamily PLA2s to be classified into two main branches: a catalytic and a myotoxic one.

Crystallization and preliminary X-ray crystallographic studies of a Lys49-phospholipase A2 homologue from Bothrops pirajai venom complexed with rosmarinic acid.

PrTX-I, a noncatalytic and myotoxic Lys49-phospholipase A(2) from Bothrops pirajai venom, was crystallized in the presence of the inhibitor rosmarinic acid (RA). This is the active compound in the methanolic extract of Cordia verbenacea, a plant that is largely used in Brazilian folk medicine. The crystals diffracted X-rays to 1.8 A resolution and the structure was solved by molecular-replacement techniques, showing electron density that corresponds to RA molecules at the entrance to the hydrophobic channel. The crystals belong to space group P2(1)2(1)2(1), indicating conformational changes in the structure after ligand binding: the crystals of all apo Lys49-phospholipase A(2) structures belong to space group P3(1)21, while the crystals of complexed structures belong to space groups P2(1) or P2(1)2(1)2(1).

Influence of quaternary conformation on the biological activities of the Asp49-phospholipases A2s from snake venoms.

One of the main components of snake venoms are the Asp49-phospholipases A(2), also known as svPLA(2)s. The study of these toxins is a matter of great scientific interest due to their wide variety of biological effects. In this work we present strong evidences found in literature and other aspects which strengthen the importance of quaternary assembly for understanding the activities and molecular evolution of svPLA(2)s.

The intriguing phospholipases A2 homologues: relevant structural features on myotoxicity and catalytic inactivity.

Phospholipases A(2) homologues are found in the venom of Crotalinae snakes, being their main action related to myonecrosis induction. Although many studies on these toxins had already been performed, their mechanism of action remains unclear. Here, important aspects about these toxins are reviewed, including their correct biological assembly and how essential is the natural substitution D49K for their catalytic inactivity.

Comparative structural studies on Lys49-phospholipases A(2) from Bothrops genus reveal their myotoxic site.

Phospholipases A(2) (PLA(2)s) are membrane-associated enzymes that hydrolyze phospholipids at the sn-2 position, releasing lysophospholipids and free fatty acids. Phospholipase A(2) homologues (Lys49-PLA(2)s) are highly myotoxic and cause extensive tissue damage despite not showing measurable catalytic activity. They are found in different snake venoms and represent one third of bothropic venom composition. The importance of these toxins during envenomation is related to the pronounced local myotoxic effect they induce since this effect is not neutralized by serum therapy. We present herein three structures of Lys49-PLA(2)s from Bothrops genus snake venom crystallized under the same conditions, two of which were grown in the presence of alpha-tocopherol (vitamin E). Comparative structural analysis of these and other Lys49-PLA(2)s showed two different patterns of oligomeric conformation that are related to the presence or absence of ligands in the hydrophobic channel. This work also confirms the biological dimer indicated by recent studies in which both C-termini are in the dimeric interface. In this configuration, we propose that the myotoxic site of these toxins is composed by the Lys 20, Lys115 and Arg118 residues. For the first time, a residue from the short-helix (Lys20) is suggested as a member of this site and the importance of Tyr119 residue to myotoxicity of bothropic Lys49-PLA(2)s is also discussed. These results support a complete hypothesis for these PLA(2)s myotoxic activity consistent with all findings on bothropic Lys49-PLA(2)s studied up to this moment, including crystallographic, bioinformatics, biochemical and biophysical data.

Structural and functional properties of Bp-LAAO, a new L-amino acid oxidase isolated from Bothrops pauloensis snake venom.

An L-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL-4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were L-Met, L-Leu, L-Phe and L-Ile and the enzyme showed a strong reduction of its catalytic activity upon L-Met and L-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAO-cDNA of 1548bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Bp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents.