Neutralization of a bothropic PLA2-like protein by caftaric acid, a novel potent inhibitor of ophidian myotoxicity.

Envenoming by snakebite is an important global health issue that has received little attention, leading the World Health Organization to naming it as neglected tropical disease. Several snakebites present serious local symptoms manifested on victims that may not be efficiently neutralized by serum therapy. Phospholipase A2-like (PLA2-like) toxins are present in Viperidae venoms and are responsible for local myotoxic activity. Herein, we investigated the association between BthTX-I toxin and caftaric acid (CFT), a molecule present in plants. CFT neutralized neuromuscular blocking and muscle-damaging activities promoted by BthTX-I. Calorimetric and light-scattering assays demonstrated that CFT inhibitor interacted with dimeric BthTX-I. Bioinformatics simulations indicated that CFT inhibitor binds to the toxin's hydrophobic channel (HCh). According to the current myotoxic mechanism, three different regions of PLA2-like toxins have specific tasks: protein allosteric activation (HCh), membrane dockage (MDoS), and membrane rupture (MDiS). We propose CFT inhibitor interferes with the allosteric activation, which is related to the conformation change leading to the exposure/alignment of MDoS/MDiS region. This is the first report of a PLA2-like toxin fully inhibited by a compound that interacts only with its HCh region. Thus, CFT is a novel candidate to complement serum therapy and improve the treatment of snakebite.

Comparative study of the interactions between fungal transcription factor nuclear localization sequences with mammalian and fungal importin-alpha.

Importin-α (Impα) is an adaptor protein that binds to cargo proteins (containing Nuclear Localization Sequences - NLSs), for their translocation to the nucleus. The specificities of the Impα/NLS interactions have been studied, since these features could be used as important tools to find potential NLSs in nuclear proteins or even for the development of targets to inhibit nuclear import or to design peptides for drug delivery. Few structural studies have compared different Impα variants from the same organism or Impα of different organisms. Previously, we investigated nuclear transport of transcription factors with Neurospora crassa Impα (NcImpα). Herein, NIT-2 and PAC-3 transcription factors NLSs were studied in complex with Mus musculus Impα (MmImpα). Calorimetric assays demonstrated that the PAC-3 NLS peptide interacts with both Impα proteins with approximately the same affinity. The NIT-2 NLS sequence binds with high affinity to the Impα major binding site from both organisms, but its binding to minor binding sites reveals interesting differences due to the presence of additional interactions of NIT-2-NLS with MmImpα. These findings, together with previous results with Impα from other organisms, indicate that the differential affinity of NLSs to minor binding sites may be also responsible for the selectivity of some cargo proteins recognition and transport.

Dual cellular localization of the Leishmania amazonensis Rbp38 (LaRbp38) explains its affinity for telomeric and mitochondrial DNA.

Rbp38 is a protein exclusively found in trypanosomatid parasites, including Leishmania amazonensis, the etiologic agent of tegumentar leishmaniasis in the Americas. The protein was first described as a Leishmania tarentolae mitochondrial RNA binding protein. Later, it was shown that the trypanosomes Rbp38 orthologues were exclusively found in the mitochondria and involved in the stabilization and replication of kinetoplast DNA (kDNA). In contrast, L. amazonensis Rbp38 (LaRbp38), co-purifies with telomerase activity and interacts not only with kDNA but also with telomeric DNA, although shares with its counterparts high sequence identity and a putative N-terminal mitochondrial targeting signal (MTS). To understand how LaRbp38 interacts both with nuclear and kDNA, we have first investigated its subcellular localization. Using hydroxy-urea synchronized L. amazonensis promastigotes we could show that LaRbp38 shuttles from mitochondria to the nucleus at late S and G2 phases. Further, we identified a non-classical nuclear localization signal (NLS) at LaRbp38 C-terminal that binds with importin alpha, a protein involved in the nuclear transport of several proteins. Also, we obtained LaRbp38 truncated forms among which, some of them also showed an affinity for both telomeric DNA and kDNA. Analysis of these truncated forms showed that LaRbp38 DNA-binding region is located between amino acid residues 95-235. Together, our findings strongly suggest that LaRbp38 is multifunctional with dual subcellular localization.

Structural basis of phospholipase A2-like myotoxin inhibition by chicoric acid, a novel potent inhibitor of ophidian toxins.

BACKGROUND: Specific compounds found in vegetal species have been demonstrated to be efficient inhibitors of snake toxins, such as phospholipase A2-like (PLA2-like) proteins. These particular proteins, present in several species of vipers (Viperidae), induce a severe local myotoxic effect in prey and human victims, and this effect is often not efficiently neutralized by the regular serum therapy. PLA2-like proteins have been functionally and structurally studied since the early 1990s; however, a comprehensive molecular mechanism was proposed only recently. METHODS: Myographic and histological techniques were used to evaluate the inhibitory effect of chicoric acid (CA) against BthTX-I myotoxin. Isothermal titration calorimetry assays were used to measure the affinity between the inhibitor and the toxin. X-ray crystallography was used to reveal details of this interaction. RESULTS: CA prevented the blockade of indirectly evoked muscle contraction and inhibited muscle damage induced by BthTX-I. The inhibitor binds to the toxin with the highest affinity measured for a natural compound in calorimetric assays. The crystal structure and molecular dynamics simulations demonstrated that CA binds at the entrance of the hydrophobic channel of the toxin and binds to one of the clusters that participates in membrane disruption. CONCLUSIONS: CA prevents the myotoxic activity of the toxin, preventing its activation by simultaneous binding with two critical regions. GENERAL SIGNIFICANCE: CA is a potential myotoxic inhibitor to other PLA2-like proteins and a possible candidate to complement serum therapy.

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.

DNA mismatch repair proteins MLH1 and PMS2 can be imported to the nucleus by a classical nuclear import pathway.

MLH1 and PMS2 proteins form the MutLα heterodimer, which plays a major role in DNA mismatch repair (MMR) in humans. Mutations in MMR-related proteins are associated with cancer, especially with colon cancer. The N-terminal region of MutLα comprises the N-termini of PMS2 and MLH1 and, similarly, the C-terminal region of MutLα is composed by the C-termini of PMS2 and MLH1, and the two are connected by linker region. The nuclear localization sequences (NLSs) necessary for the nuclear transport of the two proteins are found in this linker region. However, the exact NLS sequences have been controversial, with different sequences reported, particularly for MLH1. The individual components are not imported efficiently, presumably due to their C-termini masking their NLSs. In order to gain insights into the nuclear transport of these proteins, we solved the crystal structures of importin-α bound to peptides corresponding to the supposed NLSs of MLH1 and PMS2 and performed isothermal titration calorimetry to study their binding affinities. Both putative MLH1 and PMS2 NLSs can bind to importin-α as monopartite NLSs, which is in agreement with some previous studies. However, MLH1-NLS has the highest affinity measured by a natural NLS peptide, suggesting a major role of MLH1 protein in nuclear import compared to PMS2. Finally, the role of MLH1 and PMS2 in the nuclear transport of the MutLα heterodimer is discussed.

Nuclear transport of the Neurospora crassa NIT-2 transcription factor is mediated by importin-α.

The Neurospora crassa NIT-2 transcription factor belongs to the GATA transcription factor family and plays a fundamental role in the regulation of nitrogen metabolism. Because NIT-2 acts by accessing DNA inside the nucleus, understanding the nuclear import process of NIT-2 is necessary to characterize its function. Thus, in the present study, NIT-2 nuclear transport was investigated using a combination of biochemical, cellular, and biophysical methods. A complemented strain that produced an sfGFP-NIT-2 fusion protein was constructed, and nuclear localization assessments were made under conditions that favored protein translocation to the nucleus. Nuclear translocation was also investigated using HeLa cells, which showed that the putative NIT-2 nuclear localization sequence (NLS; 915TISSKRQRRHSKS927) was recognized by importin-α and that subsequent transport occurred via the classical import pathway. The interaction between the N. crassa importin-α (NcImpα) and the NIT-2 NLS was quantified with calorimetric assays, leading to the observation that the peptide bound to two sites with different affinities, which is typical of a monopartite NLS sequence. The crystal structure of the NcImpα/NIT-2 NLS complex was solved and revealed that the NIT-2 peptide binds to NcImpα with the major NLS-binding site playing a primary role. This result contrasts other recent studies that suggested a major role for the minor NLS-binding site in importin-α from the α2 family, indicating that both sites can be used for different cargo proteins according to specific metabolic requirements.

Biophysical studies suggest a new structural arrangement of crotoxin and provide insights into its toxic mechanism.

Crotoxin (CTX) is the main neurotoxin found in Crotalus durissus rattlesnake venoms being composed by a nontoxic and non-enzymatic component (CA) and a toxic phospholipase A2 (CB). Previous crystallographic structures of CTX and CB provided relevant insights: (i) CTX structure showed a 1:1 molecular ratio between CA and CB, presenting three tryptophan residues in the CA/CB interface and one exposed to solvent; (ii) CB structure displayed a tetrameric conformation. This study aims to provide further information on the CTX mechanism of action by several biophysical methods. Our data show that isolated CB can in fact form tetramers in solution; however, these tetramers can be dissociated by CA titration. Furthermore, CTX exhibits a strong reduction in fluorescence intensity and lifetime compared with isolated CA and CB, suggesting that all tryptophan residues in CTX may be hidden by the CA/CB interface. By companying spectroscopy fluorescence and SAXS data, we obtained a new structural model for the CTX heterodimer in which all tryptophans are located in the interface, and the N-terminal region of CB is largely exposed to the solvent. Based on this model, we propose a toxic mechanism of action for CTX, involving the interaction of N-terminal region of CB with the target before CA dissociation.

Renal ischemia/reperfusion-induced cardiac hypertrophy in mice: Cardiac morphological and morphometric characterization.

BACKGROUND: Tissue remodeling is usually dependent on the deposition of extracellular matrix that may result in tissue stiffness and impaired myocardium contraction. OBJECTIVES: We had previously demonstrated that renal ischemia/reperfusion (I/R) is able to induce development of cardiac hypertrophy in mice. Therefore, we aimed to characterize renal I/R-induced cardiac hypertrophy. DESIGN: C57BL/6 J mice were subjected to 60 minutes' unilateral renal pedicle occlusion, followed by reperfusion (I/R) for 5, 8, 12 or 15 days. Gene expression, protein abundance and morphometric analyses were performed in all time points. RESULTS: Left ventricle wall thickening was increased after eight days of reperfusion (p < 0.05). An increase in the number of heart ventricle capillaries and diameter after 12 days of reperfusion (p < 0.05) was observed; an increase in the density of capillaries starting at 5 days of reperfusion (p < 0.05) was also observed. Analyses of MMP2 protein levels showed an increase at 15 days compared to sham (p < 0.05). Moreover, TGF-ß gene expression was downregulated at 12 days as well TIMP 1 and 2 (p < 0.05). The Fourier-transform infrared spectroscopy analysis showed that collagen content was altered only in the internal section of the heart (p < 0.05); such data were supported by collagen mRNA levels. CONCLUSIONS: Renal I/R leads to impactful changes in heart morphology, accompanied by an increase in microvasculature. Although it is clear that I/R is able to induce cardiac remodeling, such morphological changes is present in only a section of the heart tissue.

Functional and structural studies of a Phospholipase A2-like protein complexed to zinc ions: Insights on its myotoxicity and inhibition mechanism.

BACKGROUND: One of the main challenges in snakebite envenomation treatment is the development of stable, versatile and efficient anti-venom therapies. Local myotoxicity in accidents involving snakes from the Bothrops genus is still a consequence of serum therapy inefficient neutralization that may lead to permanent sequelae in their victims. One of the classes of toxins that participate in muscle necrosis is the PLA2-like proteins. The aim of this work was to investigate the role of zinc ions in the inhibition of PLA2-like proteins and to advance the current knowledge of their action mechanism. METHODS: Myographic and electrophysiological techniques were used to evaluate the inhibitory effect of zinc ions, isothermal titration calorimetry assays were used to measure the affinity between zinc ions and the toxin and X-ray crystallography was used to reveal details of this interaction. RESULTS: We demonstrated that zinc ions can effectively inhibit the toxin by the interaction with two different sites, which are related to two different mechanism of inhibition: preventing membrane disruption and impairing the toxin state transition. Furthermore, structural study presented here included an additional step in the current myotoxic mechanism improving the comprehension of the allosteric transition that PLA2-like proteins undergo to exert their function. CONCLUSIONS: Our findings show that zinc ions are inhibitors of PLA2-like proteins and suggest two different mechanisms of inhibition for these ions. GENERAL SIGNIFICANCE: Zinc is a new candidate that can assist in anti-venom treatments and can promote the design of new and even more accurate structure-based inhibitors for PLA2-like proteins.

Structural and thermodynamic studies of the tobacco calmodulin-like rgs-CaM protein.

The tobacco calmodulin-like protein rgs-CaM is involved in host defense against virus and is reported to possess an associated RNA silencing suppressor activity. Rgs-CaM is also believed to act as an antiviral factor by interacting and targeting viral silencing suppressors for autophagic degradation. Despite these functional data, calcium interplay in the modulation of rgs-CaM is still poorly understood. Here we show that rgs-CaM displays a prevalent alpha-helical conformation and possesses three functional Ca2+-binding sites. Using computational modeling and molecular dynamics simulation, we demonstrate that Ca2+ binding to rgs-CaM triggers expansion of its tertiary structure with reorientation of alpha-helices within the EF-hands. This conformational change leads to the exposure of a large negatively charged region that may be implicated in the electrostatic interactions between rgs-CaM and viral suppressors. Moreover, the kd values obtained for Ca2+ binding to the three functional sites are not within the affinity range of a typical Ca2+ sensor.

Effects of photobiomodulation therapy (PBMT) on bovine sperm function.

Fertilization rates and subsequent embryo development rely on sperm factors related to semen quality and viability. Photobiomodulation therapy (PBMT) is based on emission of electromagnetic waves of a laser optical system that interact with cells and tissues resulting in biological effects. This interaction is mediated by photoacceptors that absorb the electromagnetic energy. Effects are dependent of irradiation parameters, target cell type, and species. In sperm, PBMT improves several features like motility and viability, affecting sperm aerobic metabolism and energy production. The aim of this study was to investigate, under same conditions, how different output powers (5, 7.5, and 10 mW) and time of irradiation (5 and 10 min) of laser (He-Ne laser, 633 nm) may affect frozen/thawed bovine sperm functions. Results showed significant effects depending on power while using 10 min of irradiation on motility parameters and mitochondrial potential. However, no effect was observed using 5 min of irradiation, regardless of power applied. In conclusion, PBMT is effective to modulate bovine sperm function. The effectiveness is dependent on the interaction between power applied and duration of irradiation, showing that these two parameters simultaneously influence sperm function. In this context, when using the same fluency and energy with different combinations of power and time of exposure, we observed distinct effects, revealing that biological effects should be also based on simple parameters rather than only composite parameters such as fluency, irradiance and energy. Laser irradiation of frozen/thawed bovine semen led to an increase on mitochondrial function and motility parameters that could potentially improve fertility rates.

Structural and Calorimetric Studies Demonstrate that Xeroderma Pigmentosum Type G (XPG) Can Be Imported to the Nucleus by a Classical Nuclear Import Pathway via a Monopartite NLS Sequence.

Xeroderma pigmentosum type G (XPG) proteins are involved in DNA lesion recognition and promotion of nucleotide excision repair. Specific mutations in these proteins may lead to Cockayne syndrome, in which the patients may display severe developmental retardation and neurological abnormalities. No structural information is available for their spacer region or the C-terminal domain, which are important, respectively, for specific nucleotide excision repair activity and substrate specificity, as well as nuclear translocation. Immunofluorescence studies suggested two specific regions of the XPG C-terminus as potential bipartite nuclear localization sequences, which would be responsible for its translocation to the nucleus by the classical nuclear import pathway mediated by the importin-α (Impα). Thus, in order to test these hypotheses and gain insight into the structural basis for the nuclear import process for the XPG protein, we solved the crystal structures of complexes formed by the Impα and peptides corresponding to both putative nuclear localization signal (NLS) sequences (XPG1 and XPG2) and performed isothermal titration calorimetry assays to determine their binding affinities. Structural experiments confirm the binding of both NLS peptides to Impα but, unexpectedly, they bind to the receptor as monopartite NLSs. The isothermal titration calorimetry assays demonstrated that XPG1 and XPG2 peptides bind to two separate binding sites, but with high affinity to the major NLS-binding site of the Impα, resembling classical monopartite SV40 TAg NLS. The results lead to insights about what distinguishes monopartite and bipartite NLSs, as well as the differential roles of XPG1 and XPG2 NLSs in the nuclear localization of XPG.

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.

Structure of Importin-α from a Filamentous Fungus in Complex with a Classical Nuclear Localization Signal.

Neurospora crassa is a filamentous fungus that has been extensively studied as a model organism for eukaryotic biology, providing fundamental insights into cellular processes such as cell signaling, growth and differentiation. To advance in the study of this multicellular organism, an understanding of the specific mechanisms for protein transport into the cell nucleus is essential. Importin-α (Imp-α) is the receptor for cargo proteins that contain specific nuclear localization signals (NLSs) that play a key role in the classical nuclear import pathway. Structures of Imp-α from different organisms (yeast, rice, mouse, and human) have been determined, revealing that this receptor possesses a conserved structural scaffold. However, recent studies have demonstrated that the Impα mechanism of action may vary significantly for different organisms or for different isoforms from the same organism. Therefore, structural, functional, and biophysical characterization of different Impα proteins is necessary to understand the selectivity of nuclear transport. Here, we determined the first crystal structure of an Impα from a filamentous fungus which is also the highest resolution Impα structure already solved to date (1.75 Å). In addition, we performed calorimetric analysis to determine the affinity and thermodynamic parameters of the interaction between Imp-α and the classical SV40 NLS peptide. The comparison of these data with previous studies on Impα proteins led us to demonstrate that N. crassa Imp-α possess specific features that are distinct from mammalian Imp-α but exhibit important similarities to rice Imp-α, particularly at the minor NLS binding site.